Proteomics Using Thermo Scientific Orbitrap Velos Pro...

Proteomics Applications Using Thermo Scientific

Orbitrap Velos Pro and Elite Technology

Julian Saba, Ph. D.

Proteomics Marketing

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New Ion Trap Hybrids Line up

Velos Pro

Velos Pro Orbitrap

Orbitrap Elite

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Orbitrap Elite: What is new?

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The “rotated quadrupole” with internal beam blocker

To prevent material from building up on the rods, the quadrupole has been rotated 450. This opens up a new line of sight for the neutral beam.

A beam blocker is now able to capture the neutral beam out side the body of the ion optics!

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Original Orbitrap and High Field Orbitrap Real Size Cutaways

Orbitrap Elite

• LTQ Orbitrap Classic/XL/Discovery/Velos(Pro) • (Q)Exactive

30 mm

20 mm

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Orbitrap Elite vs Orbitrap Velos Pro: Resolution and Scan Rate

Res Setting OT Velos Pro

Res Setting OT Elite

Transient [ms] Hz

15 48 7.7

7.5 30 96 6.9

15 60 192 4.0

30 120 384 2.3

60 240 768 1.2

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Orbitrap Elite scans 4x faster

Intact protein Analysis: Increased Resolution & Scan Speed

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Intact Protein Analysis Example: Yeast Enolase (48 kDa)

993.0 993.5 994.0 994.5 995.0m/z

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993.9770R=110221

994.1036R=108475

993.8706R=107582

994.1885R=109018993.6367

R=108853993.0619R=109083 994.4433

R=105946993.4244R=99350 994.7200

R=109283994.9536R=98949

994.0309R=44604

993.9030R=42104

994.1802R=42804

994.4540R=52804

993.5618R=47304

993.7112R=35704

994.6257R=37604

993.1357R=42304 994.8610

R=32404

Orbitrap Elite

Yeast Enolase, 48+ is baseline resolved on Orbitrap Elite

At 48+ charge state, the mass difference between isotopic

peaks is only 0.021 Th!

Resolution 240000 @ m/z 400 ASMS 2011 Poster MP560 Eugen Damoc et al.

LTQ Orbitrap Velos

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p [ ]

1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

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2764.2671

2664.4394

2868.56902577.0048

2923.7365

2981.00482494.9572

3041.4999

2375.89823106.6316

3167.88452304.1492

3238.36892203.6507

3308.20122141.6352

3459.08842083.1451 3541.8014

3713.17371999.2710

3903.46531922.8909

1812.5986

IgG Analysis: LTQ Orbitrap Velos

Orbi Velos @ 7500 R

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p [ ]

2800 2820 2840 2860 2880 2900 2920 2940 2960 2980m/z

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2812.4963

2868.5690

2923.73652818.6130

2981.0048

2874.4566

2929.7660

2863.1899

2824.3500

2975.0731

2880.71032936.0629

2807.08522915.3615

2830.3113

2887.2751

2943.60072835.8760

2857.1025

2950.8097

2969.3184

2898.5573

Glycoforms

LTQ Orbitrap Velos: Raw Spectrum zoom-in

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gG e5_ 0u_ 5 _ 5e _tube e s 0_ e g ad e t _ t # 60 6 6 08T: FTMS + p ESI sid=45.00 Full ms [1000.00-4000.00]

2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200m/z

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2815.46

2764.32

2712.06 2865.55

2667.31

2923.732621.38

2576.95

2981.072533.99

2492.48

2452.32 3043.83

2413.38

3102.662336.652269.39

3171.552021.95 2201.192113.94 3242.36

Orbitrap Elite @15000 R

IgG Analysis: Orbitrap Elite

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gG e5_ 0u_ 5 _ 5e _tube e s 0_ e g ad e t _ t # 60 6 6 08T: FTMS + p ESI sid=45.00 Full ms [1000.00-4000.00]

2700 2720 2740 2760 2780 2800 2820 2840m/z

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2764.32

2812.502712.06

2818.46

2767.26

2717.81

2809.50 2821.412770.14

2758.432709.18 2720.76

2824.332772.92

2723.442827.19

2776.04 2806.652725.972706.35 2778.93 2830.23

2755.61 2781.592731.80 2833.482783.65 2837.78

2737.41 2791.22 2844.442742.66

2804.11

Orbitrap Elite: Raw Spectrum zoom-in

Glycoforms – Δm = 162 Da

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OT Elite IgG ReSpect Deconvoluted Spectrum

Top Down Proteomics: Increased Resolution & Scan Speed

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OT Elite: Top Instrument for High-Throughput Top-Down Proteomics

Triosephosphate isomerase identified in Fraction 4

Elite_Fraction4_Multifrag_HRuscan #415 RT: 34.41 AV: 1 NL: 3.28E6T: FTMS + p NSI sid=15.00 Full ms [580.00-1200.00]

808.6 808.7 808.8 808.9 809.0 809.1 809.2 809.3 809.4m/z

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808.9511R=76804

z=?

809.0414R=78404

z=?809.0116R=78004

z=? 809.0728R=78504

z=?808.9219R=81804

z=?

808.8901R=77404

z=?

808.8576R=74704

z=?809.1611R=78404

z=?808.6457R=75504

z=?809.1941R=82904

z=?808.7987R=78204

z=?808.6741R=76504

z=?809.2858R=71304

z=?809.4310R=59304

z=?

z=33

26000 26500 27000 27500m/z

26647.1093

27611.3024

Theoretical MW: 26647.9 Da Observed MW: 26647.1 Da (30 ppm)

Intact mass analysis: single spectrum, 8 uscans, 120K resolution, 1e6 target

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Multiple Online Fragmentation Techniques to Maximize Coverage : ETD

Elite_Fraction4_Multifrag_HRuscan #416 RT: 34.47 AV: 1 NL: 1.14E5T: FTMS + p NSI sid=15.00 d sa Full ms2 [email protected] [250.00-2000.00]

400 600 800 1000 1200 1400 1600 1800 2000m/z

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678.3880R=48001

z=2

853.4744R=42201

z=1763.9210R=44501

z=2

493.2918R=55801

z=1

621.8456R=50301

z=2

1614.8546R=29800

z=31471.7859R=30600

z=4346.2223R=66501

z=11300.4401R=34200

z=4890.4992R=42100

z=31962.0487R=26700

z=3

1752.9369R=27804

z=?

ETD 5.26E-32

60K Resolution

1 spectrum, 8 uscans

17 N-terminal fragments 4 C-terminal fragments

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Multiple Online Fragmentation Techniques to Maximize Coverage: CID

Elite_Fraction4_Multifrag_HRuscan #417 RT: 34.54 AV: 1 NL: 2.12E5T: FTMS + p NSI sid=15.00 d Full ms2 [email protected] [345.00-2000.00]

400 600 800 1000 1200 1400 1600 1800 2000m/z

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642.1894R=48700

z=6

852.9782R=42600

z=4

696.7204R=46800

z=6

939.5015R=40304

z=?1010.8760R=40400

z=9603.3262R=50301

z=1

490.2410R=55601

z=1

819.9543R=41504

z=?

1073.2867R=37800

z=7

1205.5079R=34804

z=?

1478.7678R=34204

z=?

CID 1.02E-16

60K Resolution



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Elite_Fraction4_Multifrag_HRuscan #418 RT: 34.60 AV: 1 NL: 5.26E5T: FTMS + p NSI sid=15.00 d Full ms2 [email protected] [100.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

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909.4910R=38404

z=?

852.7272R=42200

z=4341.1480R=66201

z=1 490.2405R=55101

z=1

1073.2867R=37900

z=7

603.3255R=50001

z=1

770.4262R=44500

z=5

159.0918R=98201

z=1

961.5106R=38404

z=?

227.1043R=81304

z=?1136.6349R=36600

z=3

1303.6727R=33501

z=11565.9489R=27904

z=?

HCD 3.21E-26

60K Resolution



Multiple Online Fragmentation Techniques to Maximize Coverage : HCD

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Combined ETD, CID, and HCD Fragments Improves Sequence Coverage

More than sufficient fragmentation to identify the protein and determine isoform on online timescale

Triosephosphate isomerase

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Work Flow for Top Down Proteomics

High-throughput search using ProSightPC

yeast cell lysate

(200 – 500 μg)

reduced/ denatured

–

+

GELFrEE intact protein fractionation

10kDa

25kDa

Keys to this sample preparation: • Samples are free of contaminants which affect the success of MS experiments • Samples are separated by molecular weight – this permits developing MS acquisition methods specific to the size of the proteins in each fraction •Orbitrap Elite is the first mass spectrometer that makes high-throughput top-down proteomics possible.

Sample clean up (SDS removal)

ASMS 2011, TP560 Shannon Eliuk et al

Quantitative proteomics: Increased Accuracy & Precision

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• Quantitative cellular proteomics - SILAC

− Improved MS resolution without loss in cycle time for improved detection of co-eluting isobars for more accurate precursor-based quantitation.

Orbitrap Elite : New Standard for Proteomics Performance

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SILAC Quantification: Resolution vs. Accuracy

>100,000 resolution = distinct

30,000 resolution = indistinct

Distinct XICs for high-specificity quantitation ±2 ppm

-6 -4 -2 0 2 4 6 8

1E+3 1E+4 1E+5 1E+6 1E+7 1E+8 1E+9

Log2

(H/L

)

Area Light

Log2(H/L) vs. Area L

Median …

B.

Four orders of magnitude for 1:10 ratio

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0

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200

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1 3 5 7 9 11 13 15 17 19 ratios H:L

120 K Resolution

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200

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1 3 5 7 9 11 13 15 17 19 ratios H:L

60 K Resolution

+10% more proteins quantified

SILAC Quantification: Resolution vs. Accuracy

HeLa cell labeled with R10&K8, mixed in H:L=10:1

OT Elite :1x FTMS + 20 x ddrCID,

Protein ID results

Sample: 1ug HeLa lysate; LysC digest; 90 min gradient

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• Quantitative cellular proteomics - isobaric labeling

− Faster HCD scan rate for increased number of identified peptides in isobaric labeling experiments.

Orbitrap Elite : New Standard for Proteomics Performance

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TMT- Tandem Mass Tags

• A family of amine reactive isobaric MS/MS tags based on an identical chemical structure

*

* 127 Da

126 Da

TMT2 - Two Plex Quantitation TMT6 - Six Plex Quantitation

* * * * *

*

* *

* *

* *

* * *

* * * *

*

*

* *

* *

* * * * *

126 Da 129 Da

127 Da 130 Da

131 Da 128 Da

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TMT- Tandem Mass Tags- Reporter based quantification

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Sample Load

Orbitrap Elite LTQ Orbitrap Velos

# proteins # unique peptides

# quantified peptides # proteins # uniqued

peptides # quantified

peptides

20 ng 703 4032 4805 563 3495 3993

80 ng 873 5078 5888 694 4309 5006

500 ng 939 6531 7517 782 4853 5978

+25%

Identification data shown for 1%FDR, mean of 2 runs E.coli digest:

TMT quantification: Speed and Sensitivity of MS2

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Nine Protein Mix in E.coli Digest

Sample Number of unique peptides

Number of protein groups

Number of quantified peptides

E.Coli 5425*/4068** 923/703 6385/5019

9 proteins 271/254 9/9

551/448

E.coli+9 proteins

5696/4322 932/ 712

6936/5467

*Orbitrap Elite results, **Orbitrap Velos

+30%

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HCD spectra of TMT labeled E.coli peptide, 40 ng

Ion score 47, Scan time 0.20s

Ion score 50, Scan time 0.33s

OT Elite

OT Velos

Targeted Protein Quantitation Using HR/AM SIM

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a 100-amu wide SIM significantly improves both S/N and peak area, even with only half of the peptide amount loaded on the column.

Comparison of detection limits between full-scan and a SIM MS

Full MS (300-1200 amu) 20 fmol on column ELASGLSFPVGFK*

SIM MS (590 - 690 amu) 10 fmol on column ELASGLSFPVGFK*

Area 102 851 185 S/N = 37

Area 254 577 118 S/N = 94

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Targeted Peptide Quantitation Workflow on an Orbitrap Elite/Velos Pro Orbitrap Instrument

Multiple HR/AM SIM scans which have 100 – 200 amu isolation windows are used for quantitation of targeted

peptides.

An additional data independent ms/ms scan

using time scheduled global ms/ms list is used

for simultaneously targeted peptide verification in one

HPLC MS run. √

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OT Elite Provides More Scan Points Across Peak

Res: 60000

Res: 30000

9 scans across the peak for quantitation

5 scans across the peak for quantitation

Orbitrap Elite

LTQ Orbitrap Velos

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Comparison of analytical precision Orbitrap Elite vs LTQ Orbitrap Velos

All the targeted peptides had CVs below 15% and 96% of the targeted peptides had CVs below 10%.

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Dilution Curve for Peptide SSAAPPPPPR

The LOD for the spiked isotopically labeled peptides was 10 attomole and showed four orders of the linear

dynamic range with good analytical precision.

PTM Analysis:

Complementary Fragmentation Techniques

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Why Are Glycopeptides So Difficult To Analyze By Mass Spectrometry?

• Conventional CAD preferentially cleaves glycosidic bonds or peptide-glycan linkages

• Modifications are often highly heterogeneous • May dilute the peptide signal compared to nonglycosylated peptides

CAD Hex5HexNAc4Neu5Ac2

P

Neu5Ac Hex2HexNAc

HexHexNAc

HexHexNAcNeu5Ac

Hex2HexNAcNeu5Ac

P-HexNAc+2

P-Hex2HexNAc2+2

P-Hex2HexNAc3+2

P-Hex4HexNAc3+2

P-Hex4HexNAc3Neu5Ac+2


P-Hex3HexNAc3+2


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Glycoproteomic Strategies

Pan, S. et al., MCP

Partial information

Complete information

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CAD: glycan composition identification

ETD: peptide/glycosylation site identification

Glycopeptide identification requires CAD and ETD

Hex5HexNAc4Neu5Ac2

P

Neu5Ac

HexHexNAc

Hex2HexNAc

HexHexNAcNeu5Ac

Hex2HexNAcNeu5Ac


P-HexNAc+2



P-Hex2HexNAc2+2

P-Hex3HexNAc3+2


Serotransferrin T421-433

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Countless Possibilities for PTM analysis

Instrument Fragmentation

Method Acquisition

Method

LTQ Family CID/PQD MSn NL MS3, MSA

LTQ ETD Family + ETD

+NL ETD, CID/ETD, DDDT

Orbitrap ETD Family + HCD

+HCD/ETD, NL(product)

ETD

‘Pick and Mix’ method setup

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Discovery of Glycopeptides

Traditional approaches: • In source decay- Pseudo-MS3 of Oxonium ions Peterman, S. et al., JASMS (2006) 17, 168-179 • Low mass oxonium ions from HCD(PQD) MS/MS Spectra Snovida, S. et al., Carbohydr Res. (2010) 345, 792-801 Kuster, B. et al., JASMS (2011) 22, 931-942 Glycopeptide’s precursors identified in a post-acquisition fashion !

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Jebanathirajah, J. et al., JASMS (2003) 14, 777–784

Huddleston, M.J. et al., Analytical Chem. (1993) 65, 877-874.

The Utility of Oxonium Ions – Importance of Mass Accuracy

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Our Approach- On the Fly Identification of Glycopeptides

HCD Accurate Mass Product Dependent ETD (HCD-PD-ETD)

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Our Approach- On the Fly Identification of Glycopeptides

• Streamlines data analysis • Improves dynamic range and duty cycle

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• High Mass Accuracy MS2

• HexNAc oxonium ion is m/z 204.0866 • a2 QC ion is m/z 204.0801 • Δm/z = 32 ppm

• High Resolution MS2

• Orbitrap Elite with MS2 R = 15,000 @m/z 400

Hahne, H. & Kuster,B., JASMS (2011) 22, 931-942

The importance of resolving power

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0 10 20 30 40 50 60 70 80 90 100 110 120 Time (min) 0

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47.22 425.287

55.74 498.311 34.05

317.277 47.85 672.879 57.23

578.836 32.03 369.214 75.13

736.982 62.83

761.094 26.75 303.261 78.58

811.488 105.65 306.292 85.47

613.885

111.47 338.344

88.68 599.978

1.94 525.379

25.27 444.246

19.96 301.687

105.06 732.104

124.15 408.791

40.11 204.084

34.69 204.083

40.67 204.088

48.51 204.085 89.36

204.088

CNFCNAVVESNGTLTLSHFGK

dHex2Hex7HexNAc6Neu5Ac2

Looking for needle in a haystack – Targeting low-abundance glycopeptides

Unenriched 12 protein mixture digest

QNGTLSK

Hex4HexNAc4Neu5AcNeu5Gc dHexHex4HexNAc4Neu5Gc2

Ovomucoid

bAGP

XIC for m/z 204.087 from HCD spectra

Base Peak Chromatogram

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Looking for needle in a haystack – Targeting low-abundance glycopeptides

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HCD/ETD HCD-PD-ETD0

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# of

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HCD/ETD HCD-PD-ETD0

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HCD/ETD HCD-PD-ETD

Total number of ETD Spectra acquired

Number of Glycopeptides

identified Percentage of ETD spectra identified

160

224

SAX enriched human serum glycopeptides

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Multiple Types of Fragmentation: 50% More Unique Peptides for HCD-CID Combination

Sample: 200 ng HeLa lysate; tryptic digest; 90 min gradient.

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Phopshopeptide Analysis – ABRF 2010 sPGR study

Total # of phosphopeptides identified 21 out of 23, 0% FP, no enrichment, Instruments: LTQ Velos ETD and LTQ Velos Orbitrap ETD

3 2 16

15 15 12

ETD

CID

HCD

Phosphosites/peptide 1 2 3 4 # of peptides in study 14 5 3 1

Best result was 22 correctly identified phosphopeptides out of 23 with enrichment using LTQ Orbitrap Velos

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• Glycoproteomics is the study of the profile of glycosylated proteins, i.e. the glycoproteome, in a biological system

• Glycomics is the study of glycome (repertoire of glycans)

Glycomics vs Glycoproteomics

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Sets of MSn Spectra acquired on Velos Pro

• Glycan structural isomers differentiated using combination of permethylation, MSn and SimGlycan™ • Results shown in next 3 slides, 3 different structural isomers differentiated

1

2

1

3

2

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Permethylated Ovalbumin Glycans Identified

Structural isomers

differentiated using MSn

• Available through Premier BioSoft

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Orbitrap Elite: Summary

• The best for intact protein analysis and top-down performance • Highest resolution, 240K • Highest sensitivity (i.e. ion trap with SRIG: 5-10X vs. LTQ) • Fastest ion trap scan rate with Rapid scan (up to 7 Hz) • Fastest HCD on a hybrid instrument (5-6 Hz) • Full flexibility of fragmentation techniques (CID, HCD, ETD)

• Widest range of proteomics applications

• Top down (Intact protein analysis) • Bottom up (Protein ID) • PTMs (especially those requiring MSn such as

glycomics/glycoproteomics ) • Comprehensive Quan (SILAC, label free, targeted peptide quan with

HRAM) • Comprehensive application software suites

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Acknowledgments

Thermo Fisher Scientific, San Jose, CA San Jose Proteomics Marketing Team Bremen Orbitrap Elite R&D Team San Jose Velos Pro R&D Team Thermo Fisher Pierce, Rockford, IL

Proteomics Using Thermo Scientific Orbitrap Velos Pro...

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