b₅₊-NH₃

498.17874

y₆₊-H₂O

758.45410

y₉₊

1120.55273

y₂₊

322.18576

y₈₊

1006.51752

y₆₊

776.46594

y₁₀₊

1177.57886

y₁₊

175.11826

y₇₊

891.48932

y₅₊

647.42157

y₃₊

435.26990

y₄₊

548.35284

200 400 600 800 1000 1200

m/z

0

10

20

30

40

50

Inte

ns

ity [co

un

ts] (1

0^3

)

Extracted from: C:\Xcalibur\data\Zhiqi\ID_QE\20110321_10ngYeastDigest_Top10_140min_01.raw #23048 RT: 77.42 FTMS, HCD, z=+2, Mono m/z=645.83948 Da, MH+=1290.67168 Da, Match Tol.=20 mmu

Q Exactive – A True Qual-Quan HR/AM Mass Spectrometer for Routine Discovery and Target Quantification in Proteomics

Yi Zhang1, Zhiqi Hao1, Rosa Viner1, Shannon Eliuk1, Justin D. Blethrow1, Vlad Zabrouskov1, Markus Kellmann2 and Andreas F. Huhmer1

Thermo Fisher Scientific, San Jose, CA 95134, USA1; Thermo Fisher Scientific, Hanna-Kunath-Str. 11, 28199 Bremen, Germany2

Conclusion The Q Exactive is well suited for routine in-depth proteome analysis with its five

orders of magnitude dynamic range, fast high-resolution MS/MS scan rate of

12Hz, and high quality HR/AM HCD spectrum.

The Q Exactive generates accurate quantification data with high precision

(CV<15%) in TMT based quantitative discovery analyses.

The quadruple-based high-resolution SIM scans combined with the unique

spectrum multiplexing functionality enable high throughput HR/AM quantification

with high selectivity and sensitivity in target verification/quantification studies.

The Q Exactive is a true Qual-Quan mass spectrometer, which allows seamless

transition from discovery to target verification/quantification.

References1. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O’Shea EK, Weissman JS.

Nature 2003,16, 737–7341.

AcknowledgementsWe would like to thank Professor Gygi from Harvard University for supplying the

purified yeast digest sample.

OverviewPurpose: The performance of a novel Q Exactive mass spectrometer was evaluated in

both routine peptide identification and targeted peptide quantification.

Methods: The peptide identification capability was investigated through in-depth

analysis of the yeast proteome. Discovery-based quantification accuracy and precision

were evaluated using TMT labeled E. coli digests. The sensitivity and linear dynamic

range for HR/AM targeted quantification were investigated with peptide standards

spiked into a complex yeast digest background.

Results: The Q Exactive identified twice as many peptides/proteins as the AB Sciex

TripleTOF 5600 in a well controlled head-to-head comparison. High precision

(CV<13%) was routinely achieved for TMT-based quantification experiments on the Q

Exactive. The quadrupole based SIM and ultra-high resolution of 140K resulted in

accurate and sensitive HR/AM targeted quantification. A LOD of 10 amol was obtained

on most peptide targets with low background.

FIGURE 1. Schematic of the Q Exactive

MethodsSamples: heavy isotope labeled peptide retention standards (Thermo Fisher Scientific, Cat #:

88321), yeast tryptic digest, TMT 6-plex labeled E. coli tryptic digest.

LC: Split-free EASY-nLC from Proxeon, Solvent A: 0.1% formic acid in H2O, Solvent B: 0.1%

formic acid in acetonitrile.

Head-to-Head Comparison of the Q Exactive against the AB SciexTripleTOF 5600 for

Peptide Identification: Yeast tryptic digests at 10 ng, 100 ng and 1000 ng were analyzed with

the same nano LC column (Dionex AcclaimPepMap100 C18, 75µm x 15cm, 3µm particle,

100Å pore size) and the same 60 min gradient on both the Q Exactive and TripleTOF 5600.

The instrument parameters on the TripleTOF 5600 were set at values recommended by AB

Sciex: 250 ms full scan with 30K resolution, top 20 MS/MS with 15K resolution and 50 ms

beam time, MS2 trigger threshold of 100 cps, dynamic exclusion of 30 s. On the Q Exactive,

the resolution was 70K for full scan and 35K for MS2, the AGC target was 1E6 for full scan and

1E5 for MS2, the maximum IT was 100 ms for both full scan and MS2 scan, top 10 HCD was

selected with MS2 trigger threshold of 5,000 and dynamic exclusion of 60s.

TMT Quantification Evaluation: E. coli tryptic digests were labeled with TMT 6-plex reagents

at a ratio of 1:1:1:1:1:1. Labeled peptides (20, 40, 80, 200, 500 ng) were separated over a

Michrom Magic C18 nano LC column (75µm x 20cm, 3µm particle) with a two-hour gradient,

analyzed with a data dependent top 10 HCD method on the Q Exactive. The resolution was

70K for full scan and 17.5K for MS2. The maximum IT was 250 ms for both full scan and MS2

scan, top 10 HCD was selected with MS2 trigger threshold of 1E5 and dynamic exclusion of

80s. The AGC targets were the same as above.

HR/AM Targeted Quantification: Heavy isotope labeled peptide retention standards at 0, 10

amol, 50 amol, 100 amol, 1fmol, 10 fmol, and 100 fmol were spiked into either 10 ng or 1000

ng of yeast tryptic digest. Each sample was analyzed three times with a 60 min LC gradient

over a Michrom Magic C18 nano LC column (75µm x 15cm, 3µm particle) and a full-msx tSIM

method on the Q Exactive. The resolution was set at 140k for both scan types. The AGC

target was 1E6 for full scan and 5E5 for SIM scan. The maximum ion injection time was 100

ms for full scan and 500 ms for SIM scan. The isolation width for SIM scan was 4 amu. The

peptide retention standards were monitored over a 4 min window. Multiplexing level was set at

4, which allowed isolation and accumulation of up to four peptide targets in the c-trap before

they were transferred to Orbitrap for detection (Figure 7)

Data Analysis Peptide identification and TMT quantification data were processed and

searched against their respective databases with Mascot using standard search parameter

settings in Proteome Discoverer 1.3. Targeted quantification using LC peak areas of heavy

peptide standards with ±5 ppm mass windows were automatically calculated for both full scan

and SIM scan using Quan browser in Xcalibur 2.2. Linear regression fittings in both normal

scale and log scale were performed to estimate LOD and LOQ.

Capillary

S-lens

(Improved Sensitivity)

Bent

Flatapole

Quadrupole Mass Filter

(dd HCD, SIM)

HCD/C-Trap Combo Cell

Spectrum Multiplexing

Orbitrap with

Enhance Resolution

Discovery- Protein Identification

A Head-to-Head Comparison against TripleTOF 5600

Same sample, Same column, Same gradient

Discovery-based HR/AM Qual/Quan

High Precision, High Accuracy TMT Quantification

HR/AM Target Verification/Quantification

High Selectivity, High Sensitivity, High Throughput

Q Exactive TripleTOF 5600

Most Intense Least Intense

10 ng load

TripleTOF 5600

1000 ng load

Q Exactive

Normalized Precursor Intensity of Identified Yeast Peptides

Figure 2: Q Exactive has 10x broader dynamic range of

identification

1000ng 100ng 10ng

Figure 3: Q Exactive identified 2x unique peptides and

proteins and 4x lower abundant proteins than the

TripleTOF due to its broader dynamic range, fast scan

rate, and high quality HCD spectra.

# o

f U

niq

ue

Pe

pti

de

s#

of

Pro

tein

Gro

up

s

# o

f P

rote

in G

rou

ps

Copy Number of Yeast Proteins1

149 copy/cell in 10 ng sample

MASCOT Score: 90

L G N D D E V I L F RL G N D D E V I L F R

High Quality HCD Spectrum identified with

the Q Exactive only

In-Depth Proteome Analysis

Figure 4: TMT 6-plex Quantitative Results on Q Exactive

Amount of TMT 6-plex labeled E. coli digest

A. Quantifiable MS2 spectra account for 97%

of the total identified MS2.

B. Measured ratios are within 5% of

expected values, with CV<13%.

Ratio

1.04 1.02 1.03 1.05 1.03

TMT channels for Quantification (80ng)

Figure 5: High resolution ensures accurate target selection

30 ppm

Figure 6: High sensitivity with quadrupole based SIM scan

Figure 7: Spectrum multiplexing and concurrent

injection/detection provides high throughput analyses

Orbitrap FTMS acquisition scan 2

A

G

COrbitrap FTMS acquisition scan 1

A

G

C

Collecting ions for scan 2 Collecting ions for scan 3

Figure 8: LOD of 10 amol or 100 amol is routinely

achieved with HR/AM targeted quantification in

medium or strong background, respectively

• Low amol detection

• CV < 10% at 50 amol

• 4 orders of linearity

Sample amount (fmol)

Pe

ak a

rea

SSAAPPPPPR*

GISNEGQNASIK*

DIPVPKPK*

R2: 0.9991-0.9999

Log

10(P

eak A

rea)

Log10(Sample Amount)

10amole 1fmole 100fmole

S/N: >40

Pe

ak a

rea

R2: 0.9997-1.0000

NGFILDGFPR*

GLILVGGYGTR*

Log

10(P

eak A

rea)

Log10(Sample Amount) 10amole 1fmole 100fmole

S/N: 2.5-6

• 100 amol - 1fmol detection

• 3-4 orders of linearity

Sample amount (fmol)

A. Sensitivity and linear dynamic range of peptide targets in 10 ng yeast digest

B. Sensitivity and linear dynamic range of peptide targets in 1000 ng yeast digest

Discovery- Protein Identification

High Dynamic Range, High Sensitivity, High Duty Cycle

Amount of Yeast Tryptic Digest Loaded

1000ng 100ng 10ng