Top-Down, High-Throughput of Thermo-Stable Allergens Using Complementary MS/MS Fragmentation Strategies Monica Carrera,2 Chad Weisbrod,1 Daniel Lopez-Ferrer,1 Romain Huguet,1 Jose Manuel Gallardo,2 Jae Schwartz1 and Andreas Huhmer1 1Thermo Fisher Scientifi c, San Jose, USA; 2Instituto de Investigaciones Marinas, Vigo, Spain

Po

ster No

te 64

48

8

865 870 875 880

871.91 z=13

872.99 z=13

z=13 875.98

m/z

Top-Down, High-Throughput of Thermo-Stable Allergens Using Complementary MS/MS Fragmentation Strategies Monica Carrera2, Chad Weisbrod1, Daniel Lopez-Ferrer1, Romain Huguet1, Jose Manuel Gallardo2, Jae Schwartz1 and Andreas Huhmer1

1 Thermo Fisher Scientific, San Jose, USA. 2Instituto de Investigaciones Marinas, Vigo, Spain.

Conclusion An easy and robust method for fish speciation has been developed utilizing the high speed, high resolution and fragmentation capabilities of the Orbitrap Fusion Mass Spectrometry. Using parvalbumin proteoforms as a signature for the species identification reveals the following benefits:

We show that the sequence coverage achieved using UVPD dramatically outperforms the other fragmentation techniques

Minimal sample preparation

High sensitivity and throughput

Bypass extensive de novo sequencing due to the high homology among the amino acid sequences from the different species.

References 1.Carrera M, Cañas B, Gallardo JM J Proteomics. 2013 ;78:211-20

2.Carrera M, Cañas B, Gallardo JM J Proteomics. 2012 ;75:3211-20

3.Brunner A.M et al. Analytical Chemistry 2015 87 (8), 4152-4158

Overview Purpose: Development of a simple and fast strategy based on ultra high mass resolution that allows simultaneously the identification of food allergens and the authentication of fish species. This technique could be widely expanded and applied to determine fish species and as a tool to enforce the guidelines from the U.S. Food and Drug Administration with respect to fish labeling.

Methods: We used a two-step workflow. First, the fraction of thermo-stable proteins from the tissue was purified, and second, direct infusion using an OrbitrapTM Fusion

Results: We have developed an analytical strategy that allows to identify the presence of fish allergens and it also provides fish traceability in minutes using a high throughput LC-MS platform and top-down proteomics. These methods are the fastest in the allergen and food identification arena.

Introduction LC/MS/MS provides an accurate and sensitive way to test for allergens in food products. However, in the case of fish allergens, it is not only important to identify the allergen, but also determine the species that belongs to a given sample in order to asses traceability. Here, we present a protein based method that identifies common commercially available fish species in minutes using a simple protein extraction protocol couple to multiplex top-down proteomics using an Orbitrap Fusion Tribrid mass spectrometer modified with an UVPD source. The high mass accuracy and resolution, and the different fragmentation modes allow the classification of proteins with high protein sequence homology, but species dependent amino acid substitutions.

Methods

Results cont’

FIGURE 1. General overview of the analytical workflow. Reference muscle samples are processed. The thermo-stable proteins, beta-parvalbumins are purified and analyzed by top-down mass spectrometry

Sample Preparation

Hake was employed in this study. These were purchased at local fish markets in Vigo spain. ~1g of fresh tissue was homogenized in ~1mL of water, centrifuged and supernatants were heated at 70ºC for 5 minutes. The samples were then centrifuged at 10,000g. Supernatant with the purified parvalbumin fraction were desalted using a MSPacTM DS-10 desalting cartridge . Proteins were eluted in 80% ACN 0.1% FA and direct infused into the mass spectrometer.

Mass Spectrometry

All data was acquired on a Thermo Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). A 193-nm ArF excimer laser (Coherent ExciStar XS) was coupled to the mass spectrometer to allow for UVPD fragmentation. For direct infusion, desalted samples were direct infused in 80% ACN, 0.1% FA. A 500 μL Hamilton syringe was used for direct infusion, and the flow rate was set to 2 μL/min. Instrument control software Tune V 1.1 with EThcD, ETciD and UVPD as built-in methods was used. However, some of the data was processed using embedded Lua directly to the Orbitrap Fusion instruments. All data was acquired in the Orbitrap mass analyzer at a resolution of 120,000 (full width at half-maximum, fwhm) in intact protein mode (3 mTorr ion-routing multipole (IRM) pressure). Twenty scans, each consisting of 200 microscans were averaged. The most abundant proteoforms were subjected to fragmentation. Spectra for all fragmentation methods were acquired using a mass range of 150−2000 m/z. Precursor ion isolation was performed with the mass selecting quadrupole, and the isolation window was set to 1.5 m/z. In source fragmentation was used at 50V to avoid protein clustering in the gas phase. The precursor automatic gain control (AGC) target value was 5e5, maximum injection time 200 ms. For HCD, normalized collision energy (NCE) was set to 8, 12, 15, 17, 20, and 25%. ETD ion/ion reaction times (anion AGC 2e5, 200 ms maximum injection time), supplemental collisional activation (SA) in ETciD, and NCE in EThcD varied as indicated.

Data Analysis

Deconvoluted peaks were copied into the clipboard and paste into ProSight Lite v12. Intact protein spectra were deconvoluted with XTRACT with a signal-to-noise ratio (S/N) threshold of 10, a fit factor of 44%, mass range from 400 to 1600, and a remainder threshold of 25% (Thermo Scientific). Deconvoluted peaks were copy into the clipboard and paste into ProSight lite V12 (Proteomics Center of Excellence, Evanston, IL) for protein characterization

FIGURE 5. Characterization and Identification of 3 different hake species. The combination of the two parvalbumin isoforms allows for the complete differentiation of the three close related fish species. Isoform 1 allows for the unique identification of M. paradoxus, while the isoform 2 alows for the discrimination between M. gayi and M. merluccius.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

050

100

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

100

050

100150

200

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

250

# m

atch

ed fr

agm

ents

Prot

ein

Cove

race

(%)

m/z 200 400 2000

871.91

1600 1200 800

[M+13H]13+ NL: 3.14 E4 C, 7 B, 19

Y, 41

Z, 52A+, 46

X, 22

X+, 17

Y-, 26 A, 6

239 0

1020304050

# M

atch

ed Io

ns

Mass Accuracy (ppm)93% protein coverage

C, 42

B, 21Y, 5

Z, 26

94 0

5

10

15

20

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+*** [M+13H]13+

NL: 1.6 E6

64% protein coverage

C, 60

B, 5Y, 28

Z, 65158

0

10

20

30

40

# M

atch

ed Io

ns

Mass Accuracy (ppm)200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+*

[M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 1.6 E6

m/z 76% protein coverage

C, 62Z, 65 127

05

1015202530

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 3.06 E6

71% protein coverage

m/z 200 400 2000 1600 1200 800

NL: 1.1 E6 B, 5

Y, 60

65 05

10152025

# M

atch

ed Io

ns

Mass Accuracy (ppm)55% protein coverage

1 g of fresh tissue

Homogenization Centrifugation 5 min, 12,000 rmp

Purified Sarcoplasma

Centrifugation 5 min, 12,000 rmp Parvalbumin

Thermo

HCD ETD

EThcD ETciD UVPD

Results

90ºC

3,75

4,55

5,20

5,85

+

+

+

+

O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º pI

IEF

Original

FIGURE 2. a) IEF gel showing the effect of the thermal treatment in the purficiation of parvalbumins. b) Orbitrap Fusion MS data for purified parvalbumins from Merluccius merluccius. c) Zoom in on the m/z region 860 to 880 is shown, displaying ions originating from the different parvalbumins from their +13 precursor.

90˚C

for 5

min

FIGURE 3. Summary of the achieved sequence coverage for Parvalbumin beta-1 (Merluccius merluccius) based on the fragmentation of the +13 precursor ion (m/z 871.9) resulting from the different fragmentation methods and the experimental parameters that were evaluated.

FIGURE 4. Summary of the best results obtained for each fragmentation methods (star). Including Orbitra Fusion MSMS data, sequence coverage, total number of assigned fragment ions and histogram of the mass error for each of the matched ions.

FIGURE 5. Full characterization of the three different main isoforms for the European hake. The high ion transmission at isolation widths down to 1 amu not only allows for high sensitivity that allows the identification and characterization of the three main isoforms, but also allows for high sensitivity, specifically in the case of PRVB3 which is the less abundant of the three of them. Circles in the amino acid sequence indicate the different substitutions.

Workflow

HCD

ETD

ETciD

EThcD

UVPD

865 870 875 880

871.91 z=13

872.99 z=13

870.53 z=13

874.75

z=13

875.98 z=13

m/z m/z 1600 1300 1000 700

944.49 z=12 1031.53

z=11

871.91 z=13

1134.58 z=10

810.78 z=14

1258.98 z=9

1417.98 z=8 755.79

z=15

a) b) c)

93% protein coverage P86765-PRVB2_MERME

P02620-PRVB_MERME 86% protein coverage

P86766-PRVB3_MERME 84% protein coverage

Parvb 1

Parvb 2

865 870 875 880

871.91 z=13

872.99 z=13

z=13 875.98

m/z

Top-Down, High-Throughput of Thermo-Stable Allergens Using Complementary MS/MS Fragmentation Strategies Monica Carrera2, Chad Weisbrod1, Daniel Lopez-Ferrer1, Romain Huguet1, Jose Manuel Gallardo2, Jae Schwartz1 and Andreas Huhmer1

1 Thermo Fisher Scientific, San Jose, USA. 2Instituto de Investigaciones Marinas, Vigo, Spain.

Conclusion An easy and robust method for fish speciation has been developed utilizing the high speed, high resolution and fragmentation capabilities of the Orbitrap Fusion Mass Spectrometry. Using parvalbumin proteoforms as a signature for the species identification reveals the following benefits:

We show that the sequence coverage achieved using UVPD dramatically outperforms the other fragmentation techniques

Minimal sample preparation

High sensitivity and throughput

Bypass extensive de novo sequencing due to the high homology among the amino acid sequences from the different species.

References 1.Carrera M, Cañas B, Gallardo JM J Proteomics. 2013 ;78:211-20

2.Carrera M, Cañas B, Gallardo JM J Proteomics. 2012 ;75:3211-20

3.Brunner A.M et al. Analytical Chemistry 2015 87 (8), 4152-4158

Overview Purpose: Development of a simple and fast strategy based on ultra high mass resolution that allows simultaneously the identification of food allergens and the authentication of fish species. This technique could be widely expanded and applied to determine fish species and as a tool to enforce the guidelines from the U.S. Food and Drug Administration with respect to fish labeling.

Methods: We used a two-step workflow. First, the fraction of thermo-stable proteins from the tissue was purified, and second, direct infusion using an OrbitrapTM Fusion

Results: We have developed an analytical strategy that allows to identify the presence of fish allergens and it also provides fish traceability in minutes using a high throughput LC-MS platform and top-down proteomics. These methods are the fastest in the allergen and food identification arena.

Introduction LC/MS/MS provides an accurate and sensitive way to test for allergens in food products. However, in the case of fish allergens, it is not only important to identify the allergen, but also determine the species that belongs to a given sample in order to asses traceability. Here, we present a protein based method that identifies common commercially available fish species in minutes using a simple protein extraction protocol couple to multiplex top-down proteomics using an Orbitrap Fusion Tribrid mass spectrometer modified with an UVPD source. The high mass accuracy and resolution, and the different fragmentation modes allow the classification of proteins with high protein sequence homology, but species dependent amino acid substitutions.

Methods

Results cont’

FIGURE 1. General overview of the analytical workflow. Reference muscle samples are processed. The thermo-stable proteins, beta-parvalbumins are purified and analyzed by top-down mass spectrometry

Sample Preparation

Hake was employed in this study. These were purchased at local fish markets in Vigo spain. ~1g of fresh tissue was homogenized in ~1mL of water, centrifuged and supernatants were heated at 70ºC for 5 minutes. The samples were then centrifuged at 10,000g. Supernatant with the purified parvalbumin fraction were desalted using a MSPacTM DS-10 desalting cartridge . Proteins were eluted in 80% ACN 0.1% FA and direct infused into the mass spectrometer.

Mass Spectrometry

All data was acquired on a Thermo Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). A 193-nm ArF excimer laser (Coherent ExciStar XS) was coupled to the mass spectrometer to allow for UVPD fragmentation. For direct infusion, desalted samples were direct infused in 80% ACN, 0.1% FA. A 500 μL Hamilton syringe was used for direct infusion, and the flow rate was set to 2 μL/min. Instrument control software Tune V 1.1 with EThcD, ETciD and UVPD as built-in methods was used. However, some of the data was processed using embedded Lua directly to the Orbitrap Fusion instruments. All data was acquired in the Orbitrap mass analyzer at a resolution of 120,000 (full width at half-maximum, fwhm) in intact protein mode (3 mTorr ion-routing multipole (IRM) pressure). Twenty scans, each consisting of 200 microscans were averaged. The most abundant proteoforms were subjected to fragmentation. Spectra for all fragmentation methods were acquired using a mass range of 150−2000 m/z. Precursor ion isolation was performed with the mass selecting quadrupole, and the isolation window was set to 1.5 m/z. In source fragmentation was used at 50V to avoid protein clustering in the gas phase. The precursor automatic gain control (AGC) target value was 5e5, maximum injection time 200 ms. For HCD, normalized collision energy (NCE) was set to 8, 12, 15, 17, 20, and 25%. ETD ion/ion reaction times (anion AGC 2e5, 200 ms maximum injection time), supplemental collisional activation (SA) in ETciD, and NCE in EThcD varied as indicated.

Data Analysis

Deconvoluted peaks were copied into the clipboard and paste into ProSight Lite v12. Intact protein spectra were deconvoluted with XTRACT with a signal-to-noise ratio (S/N) threshold of 10, a fit factor of 44%, mass range from 400 to 1600, and a remainder threshold of 25% (Thermo Scientific). Deconvoluted peaks were copy into the clipboard and paste into ProSight lite V12 (Proteomics Center of Excellence, Evanston, IL) for protein characterization

FIGURE 5. Characterization and Identification of 3 different hake species. The combination of the two parvalbumin isoforms allows for the complete differentiation of the three close related fish species. Isoform 1 allows for the unique identification of M. paradoxus, while the isoform 2 alows for the discrimination between M. gayi and M. merluccius.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

050

100

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

100

050

100150

200

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

250

# m

atch

ed fr

agm

ents

Prot

ein

Cove

race

(%)

m/z 200 400 2000

871.91

1600 1200 800

[M+13H]13+ NL: 3.14 E4 C, 7 B, 19

Y, 41

Z, 52A+, 46

X, 22

X+, 17

Y-, 26 A, 6

239 0

1020304050

# M

atch

ed Io

ns

Mass Accuracy (ppm)93% protein coverage

C, 42

B, 21Y, 5

Z, 26

94 0

5

10

15

20

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+*** [M+13H]13+

NL: 1.6 E6

64% protein coverage

C, 60

B, 5Y, 28

Z, 65158

0

10

20

30

40

# M

atch

ed Io

ns

Mass Accuracy (ppm)200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+*

[M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 1.6 E6

m/z 76% protein coverage

C, 62Z, 65 127

05

1015202530

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 3.06 E6

71% protein coverage

m/z 200 400 2000 1600 1200 800

NL: 1.1 E6 B, 5

Y, 60

65 05

10152025

# M

atch

ed Io

ns

Mass Accuracy (ppm)55% protein coverage

1 g of fresh tissue

Homogenization Centrifugation 5 min, 12,000 rmp

Purified Sarcoplasma

Centrifugation 5 min, 12,000 rmp Parvalbumin

Thermo

HCD ETD

EThcD ETciD UVPD

Results

90ºC

3,75

4,55

5,20

5,85

+

+

+

+

O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º pI

IEF

Original

FIGURE 2. a) IEF gel showing the effect of the thermal treatment in the purficiation of parvalbumins. b) Orbitrap Fusion MS data for purified parvalbumins from Merluccius merluccius. c) Zoom in on the m/z region 860 to 880 is shown, displaying ions originating from the different parvalbumins from their +13 precursor.

90˚C

for 5

min

FIGURE 3. Summary of the achieved sequence coverage for Parvalbumin beta-1 (Merluccius merluccius) based on the fragmentation of the +13 precursor ion (m/z 871.9) resulting from the different fragmentation methods and the experimental parameters that were evaluated.

FIGURE 4. Summary of the best results obtained for each fragmentation methods (star). Including Orbitra Fusion MSMS data, sequence coverage, total number of assigned fragment ions and histogram of the mass error for each of the matched ions.

FIGURE 5. Full characterization of the three different main isoforms for the European hake. The high ion transmission at isolation widths down to 1 amu not only allows for high sensitivity that allows the identification and characterization of the three main isoforms, but also allows for high sensitivity, specifically in the case of PRVB3 which is the less abundant of the three of them. Circles in the amino acid sequence indicate the different substitutions.

Workflow

HCD

ETD

ETciD

EThcD

UVPD

865 870 875 880

871.91 z=13

872.99 z=13

870.53 z=13

874.75

z=13

875.98 z=13

m/z m/z 1600 1300 1000 700

944.49 z=12 1031.53

z=11

871.91 z=13

1134.58 z=10

810.78 z=14

1258.98 z=9

1417.98 z=8 755.79

z=15

a) b) c)

93% protein coverage P86765-PRVB2_MERME

P02620-PRVB_MERME 86% protein coverage

P86766-PRVB3_MERME 84% protein coverage

Parvb 1

Parvb 2

2 Top-Down, High-Throughput of Thermo-Stable Allergens Using Complementary MS/MS Fragmentation Strategies

865 870 875 880

871.91 z=13

872.99 z=13

z=13 875.98

m/z

Top-Down, High-Throughput of Thermo-Stable Allergens Using Complementary MS/MS Fragmentation Strategies Monica Carrera2, Chad Weisbrod1, Daniel Lopez-Ferrer1, Romain Huguet1, Jose Manuel Gallardo2, Jae Schwartz1 and Andreas Huhmer1

1 Thermo Fisher Scientific, San Jose, USA. 2Instituto de Investigaciones Marinas, Vigo, Spain.

Conclusion An easy and robust method for fish speciation has been developed utilizing the high speed, high resolution and fragmentation capabilities of the Orbitrap Fusion Mass Spectrometry. Using parvalbumin proteoforms as a signature for the species identification reveals the following benefits:

We show that the sequence coverage achieved using UVPD dramatically outperforms the other fragmentation techniques

Minimal sample preparation

High sensitivity and throughput

Bypass extensive de novo sequencing due to the high homology among the amino acid sequences from the different species.

References 1.Carrera M, Cañas B, Gallardo JM J Proteomics. 2013 ;78:211-20

2.Carrera M, Cañas B, Gallardo JM J Proteomics. 2012 ;75:3211-20

3.Brunner A.M et al. Analytical Chemistry 2015 87 (8), 4152-4158

Overview Purpose: Development of a simple and fast strategy based on ultra high mass resolution that allows simultaneously the identification of food allergens and the authentication of fish species. This technique could be widely expanded and applied to determine fish species and as a tool to enforce the guidelines from the U.S. Food and Drug Administration with respect to fish labeling.

Methods: We used a two-step workflow. First, the fraction of thermo-stable proteins from the tissue was purified, and second, direct infusion using an OrbitrapTM Fusion

Results: We have developed an analytical strategy that allows to identify the presence of fish allergens and it also provides fish traceability in minutes using a high throughput LC-MS platform and top-down proteomics. These methods are the fastest in the allergen and food identification arena.

Introduction LC/MS/MS provides an accurate and sensitive way to test for allergens in food products. However, in the case of fish allergens, it is not only important to identify the allergen, but also determine the species that belongs to a given sample in order to asses traceability. Here, we present a protein based method that identifies common commercially available fish species in minutes using a simple protein extraction protocol couple to multiplex top-down proteomics using an Orbitrap Fusion Tribrid mass spectrometer modified with an UVPD source. The high mass accuracy and resolution, and the different fragmentation modes allow the classification of proteins with high protein sequence homology, but species dependent amino acid substitutions.

Methods

Results cont’

FIGURE 1. General overview of the analytical workflow. Reference muscle samples are processed. The thermo-stable proteins, beta-parvalbumins are purified and analyzed by top-down mass spectrometry

Sample Preparation

Hake was employed in this study. These were purchased at local fish markets in Vigo spain. ~1g of fresh tissue was homogenized in ~1mL of water, centrifuged and supernatants were heated at 70ºC for 5 minutes. The samples were then centrifuged at 10,000g. Supernatant with the purified parvalbumin fraction were desalted using a MSPacTM DS-10 desalting cartridge . Proteins were eluted in 80% ACN 0.1% FA and direct infused into the mass spectrometer.

Mass Spectrometry

All data was acquired on a Thermo Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). A 193-nm ArF excimer laser (Coherent ExciStar XS) was coupled to the mass spectrometer to allow for UVPD fragmentation. For direct infusion, desalted samples were direct infused in 80% ACN, 0.1% FA. A 500 μL Hamilton syringe was used for direct infusion, and the flow rate was set to 2 μL/min. Instrument control software Tune V 1.1 with EThcD, ETciD and UVPD as built-in methods was used. However, some of the data was processed using embedded Lua directly to the Orbitrap Fusion instruments. All data was acquired in the Orbitrap mass analyzer at a resolution of 120,000 (full width at half-maximum, fwhm) in intact protein mode (3 mTorr ion-routing multipole (IRM) pressure). Twenty scans, each consisting of 200 microscans were averaged. The most abundant proteoforms were subjected to fragmentation. Spectra for all fragmentation methods were acquired using a mass range of 150−2000 m/z. Precursor ion isolation was performed with the mass selecting quadrupole, and the isolation window was set to 1.5 m/z. In source fragmentation was used at 50V to avoid protein clustering in the gas phase. The precursor automatic gain control (AGC) target value was 5e5, maximum injection time 200 ms. For HCD, normalized collision energy (NCE) was set to 8, 12, 15, 17, 20, and 25%. ETD ion/ion reaction times (anion AGC 2e5, 200 ms maximum injection time), supplemental collisional activation (SA) in ETciD, and NCE in EThcD varied as indicated.

Data Analysis

Deconvoluted peaks were copied into the clipboard and paste into ProSight Lite v12. Intact protein spectra were deconvoluted with XTRACT with a signal-to-noise ratio (S/N) threshold of 10, a fit factor of 44%, mass range from 400 to 1600, and a remainder threshold of 25% (Thermo Scientific). Deconvoluted peaks were copy into the clipboard and paste into ProSight lite V12 (Proteomics Center of Excellence, Evanston, IL) for protein characterization

FIGURE 5. Characterization and Identification of 3 different hake species. The combination of the two parvalbumin isoforms allows for the complete differentiation of the three close related fish species. Isoform 1 allows for the unique identification of M. paradoxus, while the isoform 2 alows for the discrimination between M. gayi and M. merluccius.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

050

100

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

100

050

100150

200

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

250

# m

atch

ed fr

agm

ents

Prot

ein

Cove

race

(%)

m/z 200 400 2000

871.91

1600 1200 800

[M+13H]13+ NL: 3.14 E4 C, 7 B, 19

Y, 41

Z, 52A+, 46

X, 22

X+, 17

Y-, 26 A, 6

239 0

1020304050

# M

atch

ed Io

ns

Mass Accuracy (ppm)93% protein coverage

C, 42

B, 21Y, 5

Z, 26

94 0

5

10

15

20

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+*** [M+13H]13+

NL: 1.6 E6

64% protein coverage

C, 60

B, 5Y, 28

Z, 65158

0

10

20

30

40

# M

atch

ed Io

ns

Mass Accuracy (ppm)200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+*

[M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 1.6 E6

m/z 76% protein coverage

C, 62Z, 65 127

05

1015202530

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 3.06 E6

71% protein coverage

m/z 200 400 2000 1600 1200 800

NL: 1.1 E6 B, 5

Y, 60

65 05

10152025

# M

atch

ed Io

ns

Mass Accuracy (ppm)55% protein coverage

1 g of fresh tissue

Homogenization Centrifugation 5 min, 12,000 rmp

Purified Sarcoplasma

Centrifugation 5 min, 12,000 rmp Parvalbumin

Thermo

HCD ETD

EThcD ETciD UVPD

Results

90ºC

3,75

4,55

5,20

5,85

+

+

+

+

O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º pI

IEF

Original

FIGURE 2. a) IEF gel showing the effect of the thermal treatment in the purficiation of parvalbumins. b) Orbitrap Fusion MS data for purified parvalbumins from Merluccius merluccius. c) Zoom in on the m/z region 860 to 880 is shown, displaying ions originating from the different parvalbumins from their +13 precursor.

90˚C

for 5

min

FIGURE 3. Summary of the achieved sequence coverage for Parvalbumin beta-1 (Merluccius merluccius) based on the fragmentation of the +13 precursor ion (m/z 871.9) resulting from the different fragmentation methods and the experimental parameters that were evaluated.

FIGURE 4. Summary of the best results obtained for each fragmentation methods (star). Including Orbitra Fusion MSMS data, sequence coverage, total number of assigned fragment ions and histogram of the mass error for each of the matched ions.

FIGURE 5. Full characterization of the three different main isoforms for the European hake. The high ion transmission at isolation widths down to 1 amu not only allows for high sensitivity that allows the identification and characterization of the three main isoforms, but also allows for high sensitivity, specifically in the case of PRVB3 which is the less abundant of the three of them. Circles in the amino acid sequence indicate the different substitutions.

Workflow

HCD

ETD

ETciD

EThcD

UVPD

865 870 875 880

871.91 z=13

872.99 z=13

870.53 z=13

874.75

z=13

875.98 z=13

m/z m/z 1600 1300 1000 700

944.49 z=12 1031.53

z=11

871.91 z=13

1134.58 z=10

810.78 z=14

1258.98 z=9

1417.98 z=8 755.79

z=15

a) b) c)

93% protein coverage P86765-PRVB2_MERME

P02620-PRVB_MERME 86% protein coverage

P86766-PRVB3_MERME 84% protein coverage

Parvb 1

Parvb 2

865 870 875 880

871.91 z=13

872.99 z=13

z=13 875.98

m/z

Top-Down, High-Throughput of Thermo-Stable Allergens Using Complementary MS/MS Fragmentation Strategies Monica Carrera2, Chad Weisbrod1, Daniel Lopez-Ferrer1, Romain Huguet1, Jose Manuel Gallardo2, Jae Schwartz1 and Andreas Huhmer1

1 Thermo Fisher Scientific, San Jose, USA. 2Instituto de Investigaciones Marinas, Vigo, Spain.

Conclusion An easy and robust method for fish speciation has been developed utilizing the high speed, high resolution and fragmentation capabilities of the Orbitrap Fusion Mass Spectrometry. Using parvalbumin proteoforms as a signature for the species identification reveals the following benefits:

We show that the sequence coverage achieved using UVPD dramatically outperforms the other fragmentation techniques

Minimal sample preparation

High sensitivity and throughput

Bypass extensive de novo sequencing due to the high homology among the amino acid sequences from the different species.

References 1.Carrera M, Cañas B, Gallardo JM J Proteomics. 2013 ;78:211-20

2.Carrera M, Cañas B, Gallardo JM J Proteomics. 2012 ;75:3211-20

3.Brunner A.M et al. Analytical Chemistry 2015 87 (8), 4152-4158

Overview Purpose: Development of a simple and fast strategy based on ultra high mass resolution that allows simultaneously the identification of food allergens and the authentication of fish species. This technique could be widely expanded and applied to determine fish species and as a tool to enforce the guidelines from the U.S. Food and Drug Administration with respect to fish labeling.

Methods: We used a two-step workflow. First, the fraction of thermo-stable proteins from the tissue was purified, and second, direct infusion using an OrbitrapTM Fusion

Results: We have developed an analytical strategy that allows to identify the presence of fish allergens and it also provides fish traceability in minutes using a high throughput LC-MS platform and top-down proteomics. These methods are the fastest in the allergen and food identification arena.

Introduction LC/MS/MS provides an accurate and sensitive way to test for allergens in food products. However, in the case of fish allergens, it is not only important to identify the allergen, but also determine the species that belongs to a given sample in order to asses traceability. Here, we present a protein based method that identifies common commercially available fish species in minutes using a simple protein extraction protocol couple to multiplex top-down proteomics using an Orbitrap Fusion Tribrid mass spectrometer modified with an UVPD source. The high mass accuracy and resolution, and the different fragmentation modes allow the classification of proteins with high protein sequence homology, but species dependent amino acid substitutions.

Methods

Results cont’

FIGURE 1. General overview of the analytical workflow. Reference muscle samples are processed. The thermo-stable proteins, beta-parvalbumins are purified and analyzed by top-down mass spectrometry

Sample Preparation

Hake was employed in this study. These were purchased at local fish markets in Vigo spain. ~1g of fresh tissue was homogenized in ~1mL of water, centrifuged and supernatants were heated at 70ºC for 5 minutes. The samples were then centrifuged at 10,000g. Supernatant with the purified parvalbumin fraction were desalted using a MSPacTM DS-10 desalting cartridge . Proteins were eluted in 80% ACN 0.1% FA and direct infused into the mass spectrometer.

Mass Spectrometry

All data was acquired on a Thermo Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). A 193-nm ArF excimer laser (Coherent ExciStar XS) was coupled to the mass spectrometer to allow for UVPD fragmentation. For direct infusion, desalted samples were direct infused in 80% ACN, 0.1% FA. A 500 μL Hamilton syringe was used for direct infusion, and the flow rate was set to 2 μL/min. Instrument control software Tune V 1.1 with EThcD, ETciD and UVPD as built-in methods was used. However, some of the data was processed using embedded Lua directly to the Orbitrap Fusion instruments. All data was acquired in the Orbitrap mass analyzer at a resolution of 120,000 (full width at half-maximum, fwhm) in intact protein mode (3 mTorr ion-routing multipole (IRM) pressure). Twenty scans, each consisting of 200 microscans were averaged. The most abundant proteoforms were subjected to fragmentation. Spectra for all fragmentation methods were acquired using a mass range of 150−2000 m/z. Precursor ion isolation was performed with the mass selecting quadrupole, and the isolation window was set to 1.5 m/z. In source fragmentation was used at 50V to avoid protein clustering in the gas phase. The precursor automatic gain control (AGC) target value was 5e5, maximum injection time 200 ms. For HCD, normalized collision energy (NCE) was set to 8, 12, 15, 17, 20, and 25%. ETD ion/ion reaction times (anion AGC 2e5, 200 ms maximum injection time), supplemental collisional activation (SA) in ETciD, and NCE in EThcD varied as indicated.

Data Analysis

Deconvoluted peaks were copied into the clipboard and paste into ProSight Lite v12. Intact protein spectra were deconvoluted with XTRACT with a signal-to-noise ratio (S/N) threshold of 10, a fit factor of 44%, mass range from 400 to 1600, and a remainder threshold of 25% (Thermo Scientific). Deconvoluted peaks were copy into the clipboard and paste into ProSight lite V12 (Proteomics Center of Excellence, Evanston, IL) for protein characterization

FIGURE 5. Characterization and Identification of 3 different hake species. The combination of the two parvalbumin isoforms allows for the complete differentiation of the three close related fish species. Isoform 1 allows for the unique identification of M. paradoxus, while the isoform 2 alows for the discrimination between M. gayi and M. merluccius.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

050

100

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

100

050

100150

200

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

250

# m

atch

ed fr

agm

ents

Prot

ein

Cove

race

(%)

m/z 200 400 2000

871.91

1600 1200 800

[M+13H]13+ NL: 3.14 E4 C, 7 B, 19

Y, 41

Z, 52A+, 46

X, 22

X+, 17

Y-, 26 A, 6

239 0

1020304050

# M

atch

ed Io

ns

Mass Accuracy (ppm)93% protein coverage

C, 42

B, 21Y, 5

Z, 26

94 0

5

10

15

20

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+*** [M+13H]13+

NL: 1.6 E6

64% protein coverage

C, 60

B, 5Y, 28

Z, 65158

0

10

20

30

40

# M

atch

ed Io

ns

Mass Accuracy (ppm)200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+*

[M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 1.6 E6

m/z 76% protein coverage

C, 62Z, 65 127

05

1015202530

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 3.06 E6

71% protein coverage

m/z 200 400 2000 1600 1200 800

NL: 1.1 E6 B, 5

Y, 60

65 05

10152025

# M

atch

ed Io

ns

Mass Accuracy (ppm)55% protein coverage

1 g of fresh tissue

Homogenization Centrifugation 5 min, 12,000 rmp

Purified Sarcoplasma

Centrifugation 5 min, 12,000 rmp Parvalbumin

Thermo

HCD ETD

EThcD ETciD UVPD

Results

90ºC

3,75

4,55

5,20

5,85

+

+

+

+

O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º pI

IEF

Original

FIGURE 2. a) IEF gel showing the effect of the thermal treatment in the purficiation of parvalbumins. b) Orbitrap Fusion MS data for purified parvalbumins from Merluccius merluccius. c) Zoom in on the m/z region 860 to 880 is shown, displaying ions originating from the different parvalbumins from their +13 precursor.

90˚C

for 5

min

FIGURE 3. Summary of the achieved sequence coverage for Parvalbumin beta-1 (Merluccius merluccius) based on the fragmentation of the +13 precursor ion (m/z 871.9) resulting from the different fragmentation methods and the experimental parameters that were evaluated.

FIGURE 4. Summary of the best results obtained for each fragmentation methods (star). Including Orbitra Fusion MSMS data, sequence coverage, total number of assigned fragment ions and histogram of the mass error for each of the matched ions.

FIGURE 5. Full characterization of the three different main isoforms for the European hake. The high ion transmission at isolation widths down to 1 amu not only allows for high sensitivity that allows the identification and characterization of the three main isoforms, but also allows for high sensitivity, specifically in the case of PRVB3 which is the less abundant of the three of them. Circles in the amino acid sequence indicate the different substitutions.

Workflow

HCD

ETD

ETciD

EThcD

UVPD

865 870 875 880

871.91 z=13

872.99 z=13

870.53 z=13

874.75

z=13

875.98 z=13

m/z m/z 1600 1300 1000 700

944.49 z=12 1031.53

z=11

871.91 z=13

1134.58 z=10

810.78 z=14

1258.98 z=9

1417.98 z=8 755.79

z=15

a) b) c)

93% protein coverage P86765-PRVB2_MERME

P02620-PRVB_MERME 86% protein coverage

P86766-PRVB3_MERME 84% protein coverage

Parvb 1

Parvb 2

3Thermo Scientifi c Poster Note • ASMS • PN64888-EN 0615S

865 870 875 880

871.91 z=13

872.99 z=13

z=13 875.98

m/z

Top-Down, High-Throughput of Thermo-Stable Allergens Using Complementary MS/MS Fragmentation Strategies Monica Carrera2, Chad Weisbrod1, Daniel Lopez-Ferrer1, Romain Huguet1, Jose Manuel Gallardo2, Jae Schwartz1 and Andreas Huhmer1

1 Thermo Fisher Scientific, San Jose, USA. 2Instituto de Investigaciones Marinas, Vigo, Spain.

Conclusion An easy and robust method for fish speciation has been developed utilizing the high speed, high resolution and fragmentation capabilities of the Orbitrap Fusion Mass Spectrometry. Using parvalbumin proteoforms as a signature for the species identification reveals the following benefits:

We show that the sequence coverage achieved using UVPD dramatically outperforms the other fragmentation techniques

Minimal sample preparation

High sensitivity and throughput

Bypass extensive de novo sequencing due to the high homology among the amino acid sequences from the different species.

References 1.Carrera M, Cañas B, Gallardo JM J Proteomics. 2013 ;78:211-20

2.Carrera M, Cañas B, Gallardo JM J Proteomics. 2012 ;75:3211-20

3.Brunner A.M et al. Analytical Chemistry 2015 87 (8), 4152-4158

Overview Purpose: Development of a simple and fast strategy based on ultra high mass resolution that allows simultaneously the identification of food allergens and the authentication of fish species. This technique could be widely expanded and applied to determine fish species and as a tool to enforce the guidelines from the U.S. Food and Drug Administration with respect to fish labeling.

Methods: We used a two-step workflow. First, the fraction of thermo-stable proteins from the tissue was purified, and second, direct infusion using an OrbitrapTM Fusion

Results: We have developed an analytical strategy that allows to identify the presence of fish allergens and it also provides fish traceability in minutes using a high throughput LC-MS platform and top-down proteomics. These methods are the fastest in the allergen and food identification arena.

Introduction LC/MS/MS provides an accurate and sensitive way to test for allergens in food products. However, in the case of fish allergens, it is not only important to identify the allergen, but also determine the species that belongs to a given sample in order to asses traceability. Here, we present a protein based method that identifies common commercially available fish species in minutes using a simple protein extraction protocol couple to multiplex top-down proteomics using an Orbitrap Fusion Tribrid mass spectrometer modified with an UVPD source. The high mass accuracy and resolution, and the different fragmentation modes allow the classification of proteins with high protein sequence homology, but species dependent amino acid substitutions.

Methods

Results cont’

FIGURE 1. General overview of the analytical workflow. Reference muscle samples are processed. The thermo-stable proteins, beta-parvalbumins are purified and analyzed by top-down mass spectrometry

Sample Preparation

Hake was employed in this study. These were purchased at local fish markets in Vigo spain. ~1g of fresh tissue was homogenized in ~1mL of water, centrifuged and supernatants were heated at 70ºC for 5 minutes. The samples were then centrifuged at 10,000g. Supernatant with the purified parvalbumin fraction were desalted using a MSPacTM DS-10 desalting cartridge . Proteins were eluted in 80% ACN 0.1% FA and direct infused into the mass spectrometer.

Mass Spectrometry

All data was acquired on a Thermo Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). A 193-nm ArF excimer laser (Coherent ExciStar XS) was coupled to the mass spectrometer to allow for UVPD fragmentation. For direct infusion, desalted samples were direct infused in 80% ACN, 0.1% FA. A 500 μL Hamilton syringe was used for direct infusion, and the flow rate was set to 2 μL/min. Instrument control software Tune V 1.1 with EThcD, ETciD and UVPD as built-in methods was used. However, some of the data was processed using embedded Lua directly to the Orbitrap Fusion instruments. All data was acquired in the Orbitrap mass analyzer at a resolution of 120,000 (full width at half-maximum, fwhm) in intact protein mode (3 mTorr ion-routing multipole (IRM) pressure). Twenty scans, each consisting of 200 microscans were averaged. The most abundant proteoforms were subjected to fragmentation. Spectra for all fragmentation methods were acquired using a mass range of 150−2000 m/z. Precursor ion isolation was performed with the mass selecting quadrupole, and the isolation window was set to 1.5 m/z. In source fragmentation was used at 50V to avoid protein clustering in the gas phase. The precursor automatic gain control (AGC) target value was 5e5, maximum injection time 200 ms. For HCD, normalized collision energy (NCE) was set to 8, 12, 15, 17, 20, and 25%. ETD ion/ion reaction times (anion AGC 2e5, 200 ms maximum injection time), supplemental collisional activation (SA) in ETciD, and NCE in EThcD varied as indicated.

Data Analysis

Deconvoluted peaks were copied into the clipboard and paste into ProSight Lite v12. Intact protein spectra were deconvoluted with XTRACT with a signal-to-noise ratio (S/N) threshold of 10, a fit factor of 44%, mass range from 400 to 1600, and a remainder threshold of 25% (Thermo Scientific). Deconvoluted peaks were copy into the clipboard and paste into ProSight lite V12 (Proteomics Center of Excellence, Evanston, IL) for protein characterization

FIGURE 5. Characterization and Identification of 3 different hake species. The combination of the two parvalbumin isoforms allows for the complete differentiation of the three close related fish species. Isoform 1 allows for the unique identification of M. paradoxus, while the isoform 2 alows for the discrimination between M. gayi and M. merluccius.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

050

100

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

100

050

100150

200

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

250

# m

atch

ed fr

agm

ents

Prot

ein

Cove

race

(%)

m/z 200 400 2000

871.91

1600 1200 800

[M+13H]13+ NL: 3.14 E4 C, 7 B, 19

Y, 41

Z, 52A+, 46

X, 22

X+, 17

Y-, 26 A, 6

239 0

1020304050

# M

atch

ed Io

ns

Mass Accuracy (ppm)93% protein coverage

C, 42

B, 21Y, 5

Z, 26

94 0

5

10

15

20

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+*** [M+13H]13+

NL: 1.6 E6

64% protein coverage

C, 60

B, 5Y, 28

Z, 65158

0

10

20

30

40

# M

atch

ed Io

ns

Mass Accuracy (ppm)200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+*

[M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 1.6 E6

m/z 76% protein coverage

C, 62Z, 65 127

05

1015202530

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 3.06 E6

71% protein coverage

m/z 200 400 2000 1600 1200 800

NL: 1.1 E6 B, 5

Y, 60

65 05

10152025

# M

atch

ed Io

ns

Mass Accuracy (ppm)55% protein coverage

1 g of fresh tissue

Homogenization Centrifugation 5 min, 12,000 rmp

Purified Sarcoplasma

Centrifugation 5 min, 12,000 rmp Parvalbumin

Thermo

HCD ETD

EThcD ETciD UVPD

Results

90ºC

3,75

4,55

5,20

5,85

+

+

+

+

O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º pI

IEF

Original

FIGURE 2. a) IEF gel showing the effect of the thermal treatment in the purficiation of parvalbumins. b) Orbitrap Fusion MS data for purified parvalbumins from Merluccius merluccius. c) Zoom in on the m/z region 860 to 880 is shown, displaying ions originating from the different parvalbumins from their +13 precursor.

90˚C

for 5

min

FIGURE 3. Summary of the achieved sequence coverage for Parvalbumin beta-1 (Merluccius merluccius) based on the fragmentation of the +13 precursor ion (m/z 871.9) resulting from the different fragmentation methods and the experimental parameters that were evaluated.

FIGURE 4. Summary of the best results obtained for each fragmentation methods (star). Including Orbitra Fusion MSMS data, sequence coverage, total number of assigned fragment ions and histogram of the mass error for each of the matched ions.

FIGURE 5. Full characterization of the three different main isoforms for the European hake. The high ion transmission at isolation widths down to 1 amu not only allows for high sensitivity that allows the identification and characterization of the three main isoforms, but also allows for high sensitivity, specifically in the case of PRVB3 which is the less abundant of the three of them. Circles in the amino acid sequence indicate the different substitutions.

Workflow

HCD

ETD

ETciD

EThcD

UVPD

865 870 875 880

871.91 z=13

872.99 z=13

870.53 z=13

874.75

z=13

875.98 z=13

m/z m/z 1600 1300 1000 700

944.49 z=12 1031.53

z=11

871.91 z=13

1134.58 z=10

810.78 z=14

1258.98 z=9

1417.98 z=8 755.79

z=15

a) b) c)

93% protein coverage P86765-PRVB2_MERME

P02620-PRVB_MERME 86% protein coverage

P86766-PRVB3_MERME 84% protein coverage

Parvb 1

Parvb 2

PN64488-EN 0615S

865 870 875 880

871.91 z=13

872.99 z=13

z=13 875.98

m/z

Top-Down, High-Throughput of Thermo-Stable Allergens Using Complementary MS/MS Fragmentation Strategies Monica Carrera2, Chad Weisbrod1, Daniel Lopez-Ferrer1, Romain Huguet1, Jose Manuel Gallardo2, Jae Schwartz1 and Andreas Huhmer1

1 Thermo Fisher Scientific, San Jose, USA. 2Instituto de Investigaciones Marinas, Vigo, Spain.

Conclusion An easy and robust method for fish speciation has been developed utilizing the high speed, high resolution and fragmentation capabilities of the Orbitrap Fusion Mass Spectrometry. Using parvalbumin proteoforms as a signature for the species identification reveals the following benefits:

We show that the sequence coverage achieved using UVPD dramatically outperforms the other fragmentation techniques

Minimal sample preparation

High sensitivity and throughput

Bypass extensive de novo sequencing due to the high homology among the amino acid sequences from the different species.

References 1.Carrera M, Cañas B, Gallardo JM J Proteomics. 2013 ;78:211-20

2.Carrera M, Cañas B, Gallardo JM J Proteomics. 2012 ;75:3211-20

3.Brunner A.M et al. Analytical Chemistry 2015 87 (8), 4152-4158

Overview Purpose: Development of a simple and fast strategy based on ultra high mass resolution that allows simultaneously the identification of food allergens and the authentication of fish species. This technique could be widely expanded and applied to determine fish species and as a tool to enforce the guidelines from the U.S. Food and Drug Administration with respect to fish labeling.

Methods: We used a two-step workflow. First, the fraction of thermo-stable proteins from the tissue was purified, and second, direct infusion using an OrbitrapTM Fusion

Results: We have developed an analytical strategy that allows to identify the presence of fish allergens and it also provides fish traceability in minutes using a high throughput LC-MS platform and top-down proteomics. These methods are the fastest in the allergen and food identification arena.

Introduction LC/MS/MS provides an accurate and sensitive way to test for allergens in food products. However, in the case of fish allergens, it is not only important to identify the allergen, but also determine the species that belongs to a given sample in order to asses traceability. Here, we present a protein based method that identifies common commercially available fish species in minutes using a simple protein extraction protocol couple to multiplex top-down proteomics using an Orbitrap Fusion Tribrid mass spectrometer modified with an UVPD source. The high mass accuracy and resolution, and the different fragmentation modes allow the classification of proteins with high protein sequence homology, but species dependent amino acid substitutions.

Methods

Results cont’

FIGURE 1. General overview of the analytical workflow. Reference muscle samples are processed. The thermo-stable proteins, beta-parvalbumins are purified and analyzed by top-down mass spectrometry

Sample Preparation

Hake was employed in this study. These were purchased at local fish markets in Vigo spain. ~1g of fresh tissue was homogenized in ~1mL of water, centrifuged and supernatants were heated at 70ºC for 5 minutes. The samples were then centrifuged at 10,000g. Supernatant with the purified parvalbumin fraction were desalted using a MSPacTM DS-10 desalting cartridge . Proteins were eluted in 80% ACN 0.1% FA and direct infused into the mass spectrometer.

Mass Spectrometry

All data was acquired on a Thermo Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). A 193-nm ArF excimer laser (Coherent ExciStar XS) was coupled to the mass spectrometer to allow for UVPD fragmentation. For direct infusion, desalted samples were direct infused in 80% ACN, 0.1% FA. A 500 μL Hamilton syringe was used for direct infusion, and the flow rate was set to 2 μL/min. Instrument control software Tune V 1.1 with EThcD, ETciD and UVPD as built-in methods was used. However, some of the data was processed using embedded Lua directly to the Orbitrap Fusion instruments. All data was acquired in the Orbitrap mass analyzer at a resolution of 120,000 (full width at half-maximum, fwhm) in intact protein mode (3 mTorr ion-routing multipole (IRM) pressure). Twenty scans, each consisting of 200 microscans were averaged. The most abundant proteoforms were subjected to fragmentation. Spectra for all fragmentation methods were acquired using a mass range of 150−2000 m/z. Precursor ion isolation was performed with the mass selecting quadrupole, and the isolation window was set to 1.5 m/z. In source fragmentation was used at 50V to avoid protein clustering in the gas phase. The precursor automatic gain control (AGC) target value was 5e5, maximum injection time 200 ms. For HCD, normalized collision energy (NCE) was set to 8, 12, 15, 17, 20, and 25%. ETD ion/ion reaction times (anion AGC 2e5, 200 ms maximum injection time), supplemental collisional activation (SA) in ETciD, and NCE in EThcD varied as indicated.

Data Analysis

Deconvoluted peaks were copied into the clipboard and paste into ProSight Lite v12. Intact protein spectra were deconvoluted with XTRACT with a signal-to-noise ratio (S/N) threshold of 10, a fit factor of 44%, mass range from 400 to 1600, and a remainder threshold of 25% (Thermo Scientific). Deconvoluted peaks were copy into the clipboard and paste into ProSight lite V12 (Proteomics Center of Excellence, Evanston, IL) for protein characterization

FIGURE 5. Characterization and Identification of 3 different hake species. The combination of the two parvalbumin isoforms allows for the complete differentiation of the three close related fish species. Isoform 1 allows for the unique identification of M. paradoxus, while the isoform 2 alows for the discrimination between M. gayi and M. merluccius.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

050

100

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

100

050

100150

200

HCD_12_N

CEHCD_

15_NCE

HCD_17_N

CEHCD_

20_NCE

ETD_3_ms

ETD_4_ms

ETD_6_ms

ETD_8_ms

ETciD_4_m

s_10_SA

ETciD_4_m

s_15_SA

ETciD_6_m

s_10_SA

ETciD_6_m

s_15_SA

EThcD_4_m

s_8_NCE

EThcD_4_m

s_10_NCE

EThcD_4_m

s_12_NCE

EThcD_4_m

s_15_NCE

EThcD_6_m

s_8_NCE

EThcD_6_m

s_10_NCE

EThcD_6_m

s_12_NCE

EThcD_6_m

s_15_NCE

UVPD_0.8_

mJ_1_Puls

eUVPD

_0.8_mJ_2

_Pulse

UVPD_0.8_

mJ_3_Puls

eUVPD

_0.8_mJ_4

_Pulse

UVPD_1.3_

mJ_1_Puls

eUVPD

_1.3_mJ_2

_Pulse

UVPD_1.3_

mJ_3_Puls

eUVPD

_1.3_mJ_4

_Pulse

UVPD_1.8_

mJ_1_Puls

eUVPD

_1.8_mJ_2

_Pulse

UVPD_2.8_

mJ_1_Puls

eUVPD

_2.8_mJ_2

_Pulse 0

5010

015

020

0

HCD_

12_N

CEHC

D_15

_NCE

HCD_

17_N

CEHC

D_20

_NCE

ETD_

3_m

sET

D_4_

ms

ETD_

6_m

sET

D_8_

ms

ETci

D_4_

ms_

10_S

AET

ciD_

4_m

s_15

_SA

ETci

D_6_

ms_

10_S

AET

ciD_

6_m

s_15

_SA

EThc

D_4_

ms_

8_N

CEET

hcD_

4_m

s_10

_NCE

EThc

D_4_

ms_

12_N

CEET

hcD_

4_m

s_15

_NCE

EThc

D_6_

ms_

8_N

CEET

hcD_

6_m

s_10

_NCE

EThc

D_6_

ms_

12_N

CEET

hcD_

6_m

s_15

_NCE

UVP

D_0.

8_m

J_1_

Pulse

UVP

D_0.

8_m

J_2_

Pulse

UVP

D_0.

8_m

J_3_

Pulse

UVP

D_0.

8_m

J_4_

Pulse

UVP

D_1.

3_m

J_1_

Pulse

UVP

D_1.

3_m

J_2_

Pulse

UVP

D_1.

3_m

J_3_

Pulse

UVP

D_1.

3_m

J_4_

Pulse

UVP

D_1.

8_m

J_1_

Pulse

UVP

D_1.

8_m

J_2_

Pulse

UVP

D_2.

8_m

J_1_

Pulse

UVP

D_2.

8_m

J_2_

Pulse

0

250

# m

atch

ed fr

agm

ents

Prot

ein

Cove

race

(%)

m/z 200 400 2000

871.91

1600 1200 800

[M+13H]13+ NL: 3.14 E4 C, 7 B, 19

Y, 41

Z, 52A+, 46

X, 22

X+, 17

Y-, 26 A, 6

239 0

1020304050

# M

atch

ed Io

ns

Mass Accuracy (ppm)93% protein coverage

C, 42

B, 21Y, 5

Z, 26

94 0

5

10

15

20

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+*** [M+13H]13+

NL: 1.6 E6

64% protein coverage

C, 60

B, 5Y, 28

Z, 65158

0

10

20

30

40

# M

atch

ed Io

ns

Mass Accuracy (ppm)200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+*

[M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 1.6 E6

m/z 76% protein coverage

C, 62Z, 65 127

05

1015202530

# M

atch

ed Io

ns

Mass Accuracy (ppm)m/z 200 400 2000

871.91

944.57 1030.44

1133.38

1600 1200 800

[M+13H]12+* [M+13H]11+**

[M+13H]10+***

[M+13H]13+

NL: 3.06 E6

71% protein coverage

m/z 200 400 2000 1600 1200 800

NL: 1.1 E6 B, 5

Y, 60

65 05

10152025

# M

atch

ed Io

ns

Mass Accuracy (ppm)55% protein coverage

1 g of fresh tissue

Homogenization Centrifugation 5 min, 12,000 rmp

Purified Sarcoplasma

Centrifugation 5 min, 12,000 rmp Parvalbumin

Thermo

HCD ETD

EThcD ETciD UVPD

Results

90ºC

3,75

4,55

5,20

5,85

+

+

+

+

O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º O 60º 70º 80º 90º pI

IEF

Original

FIGURE 2. a) IEF gel showing the effect of the thermal treatment in the purficiation of parvalbumins. b) Orbitrap Fusion MS data for purified parvalbumins from Merluccius merluccius. c) Zoom in on the m/z region 860 to 880 is shown, displaying ions originating from the different parvalbumins from their +13 precursor.

90˚C

for 5

min

FIGURE 3. Summary of the achieved sequence coverage for Parvalbumin beta-1 (Merluccius merluccius) based on the fragmentation of the +13 precursor ion (m/z 871.9) resulting from the different fragmentation methods and the experimental parameters that were evaluated.

FIGURE 4. Summary of the best results obtained for each fragmentation methods (star). Including Orbitra Fusion MSMS data, sequence coverage, total number of assigned fragment ions and histogram of the mass error for each of the matched ions.

FIGURE 5. Full characterization of the three different main isoforms for the European hake. The high ion transmission at isolation widths down to 1 amu not only allows for high sensitivity that allows the identification and characterization of the three main isoforms, but also allows for high sensitivity, specifically in the case of PRVB3 which is the less abundant of the three of them. Circles in the amino acid sequence indicate the different substitutions.

Workflow

HCD

ETD

ETciD

EThcD

UVPD

865 870 875 880

871.91 z=13

872.99 z=13

870.53 z=13

874.75

z=13

875.98 z=13

m/z m/z 1600 1300 1000 700

944.49 z=12 1031.53

z=11

871.91 z=13

1134.58 z=10

810.78 z=14

1258.98 z=9

1417.98 z=8 755.79

z=15

a) b) c)

93% protein coverage P86765-PRVB2_MERME

P02620-PRVB_MERME 86% protein coverage

P86766-PRVB3_MERME 84% protein coverage

Parvb 1

Parvb 2

Africa +43 1 333 50 34 0Australia +61 3 9757 4300Austria +43 810 282 206Belgium +32 53 73 42 41Canada +1 800 530 8447China 800 810 5118 (free call domestic)

400 650 5118

Denmark +45 70 23 62 60Europe-Other +43 1 333 50 34 0Finland +358 10 3292 200France +33 1 60 92 48 00Germany +49 6103 408 1014India +91 22 6742 9494Italy +39 02 950 591

Japan +81 45 453 9100Korea +82 2 3420 8600Latin America +1 561 688 8700Middle East +43 1 333 50 34 0Netherlands +31 76 579 55 55New Zealand +64 9 980 6700Norway +46 8 556 468 00

Russia/CIS +43 1 333 50 34 0Singapore +65 6289 1190Spain +34 914 845 965Sweden +46 8 556 468 00Switzerland +41 61 716 77 00UK +44 1442 233555USA +1 800 532 4752

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