Protein separation and sample preparation for mass spectrometry - - PDF document

1

Protein separation and sample preparation for mass spectrometry

Manfredo Quadroni www.unil.ch/paf

• 3. Interfacing to MS
• 3. Interfacing to MS
• 2. Protein
• 2. Protein

Separation Separation

• 1. Protein
• 1. Protein

Pre-Fractionation Pre-Fractionation

PTM analysis PTM analysis FOCUS : Single protein 1-20 proteins Interaction / Functional Interaction / Functional Proteomics Proteomics FOCUS : Subcellular fraction Organelle Protein Complex 1-200 proteins Protein expression Protein expression analysis analysis FOCUS : Complex samples Whole proteomes 200 and more proteins

• IP (need good Ab)
• Multi-step LC
• other affinity
• Physical properties

1D-SDS PAGE 2D-PAGE MALDI - MS Protein Digestion Sample Cleanup / separation: RP-LC ESI - MS

• Frac. sedimentation
• Co-IP (complexes)
• Multi-step LC
• other affinity
• Physical properties
• Size exclusion
• Multi-step LC

fractionation Raw extract Raw extract Raw extract

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Protein separation/fractionation : a revival of old fashioned biochemistry

All “classical” methods can be used in all possible combinations : Liquid chromatography :

• anion exchange
• cation exchange
• hydrophobic interaction
• hydroxylapatite, heparin,…
• batch methods : fractional precipitations
• affinity : Immobilised Antibodies,

tagged molecules, drugs, peptides,

• size exclusion

Isoelectric focusing & chromatofocusing Electrophoresis :

• SDS-PAGE ( 1D & 2D )
• native (Urea, Blue Native, Triton Acidic Urea)
• …

Key points :

Choose methods based on orthogonal separation principles Choose methods compatible on the basis of sample composition

GEL+BUFFER LIMITED NATIVE MEDIUM SURFACE CHARGE BLUE NATIVE GEL GEL+SDS+BUFFER LIMITED DENATURING VERY HIGH NET CHARGE + SIZE 2D SDS PAGE GEL+SDS+BUFFER LIMITED DENATURING HIGH SIZE 1D-SDS PAGE BUFFER+AMPHOLYTES+ DENATURANTS MEDIUM DENATURING MEDIUM-HIGH NET CHARGE IEF BUFFER+ ADDITIVES* YES NATIVE* N.A. BINDING TO MATRIX LC:SPECIFIC AFFINITY BUFFER YES NATIVE LOW-MEDIUM SIZE LC: SIZE EXCLUSION / GEL FILTRATION BUFFER + SOLVENT YES NATIVE LOW-MEDIUM SURFACE HYDROPHOBICI TY LC : HYDROPHOBIC BUFFER + SALT YES NATIVE LOW-MEDIUM SURFACE CHARGE LC : CATION EXCHANGE BUFFER + SALT YES NATIVE LOW-MEDIUM SURFACE CHARGE LC : ANION EXCHANGE SAMPLE RECOVERY IN SCALABILITY NATIVE OR DENATURING RESOLUTION PRINCIPLE OF SEPARATION

Protein separation/fractionation : a revival of old fashioned biochemistry

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After digestion : separation of peptides

Liquid chromatography :

• strong cation exchange (SCX)
• strong anion exchange (SAX)
• reversed-phase (RP)
• immobilised metal affinity (IMAC)

SCX SAX IMAC RP MS

P-peptides separation separation Separation + desalting Protease digestion (Trypsin)

Purity separate complex mixtures Cleanliness eliminate molecules that interfere with ionisation/detection Interfacing to MS: goals Interfacing to MS: goals

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Most charged or Most charged or ionisable ionisable molecules interfere with the molecules interfere with the ionisation ionisation of the analyte (i.e. compete for charges ) and cause

• f the analyte (i.e. compete for charges ) and cause

signal suppression and/or elevation of the background noise signal suppression and/or elevation of the background noise Interfacing to MS: Interfacing to MS: “ “Killer Killer” ” substances to eliminate substances to eliminate Salts : Na+, Cl-, Tris, … Chaotropes : Urea, Thiourea, Gu-HCl, Detergents : SDS, NP-40, Triton X, TWEEN,… Polymers : PEG, Ampholytes, .. All non volatile, ionic compounds : glycerol,DMSO,…

np-40/Scans 1-36 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 100 75 50 25 Relative Intensity (%) 65,945 trial ppeptides - 1.10.1996 - 12:29 No Title +Profile Q3SCAN Average of scans 1 to 36 Time=0.55 min

JW1 Peptide, 4 mg / ml

44 Da 44 Da 44 Da 44 Da 44 Da 44 Da 44 Da 44 Da 44 Da

Interfacing to MS: signal suppression by NP-40 Interfacing to MS: signal suppression by NP-40

Solvent : 50 % MeOH, 0.5 % Formic acid

? % NP-40

Sample was “cleaned up” on C18

• > concentration of detergent rather

than peptide

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Good crystallisation (DHB) No crystallisation due to salt or detergent  no signal !!! α-CHCA DHB

MALDI: MALDI: crystallisation crystallisation is essential is essential

Reversed-phase

• Reversed-phase

Reversed-phase Reversed-phase Reversed-phase Further clean-up by

• +++

+++ +++ + ++ + ++ Applicable to peptides ++ SCX or Reversed- phase C18, C8,C4 Zwitterionic detergents (CHAPS) (+) SCX PEGs ++++ SDS-PAGE Many

• ?

Ampholytes + Reversed-phase C18, C8,C4 Buffers + Reversed-phase C18, C8,C4 Chaotropes (Urea) + Reversed-phase C18, C8,C4 Salts (+) SCX Non ionic detergents (NP- 40, TWEEN, Triton x100) + HILIC SCX SDS Applicable to proteins Technique Molecule to eliminate

Liquid samples: removal of Liquid samples: removal of ‘ ‘contaminants contaminants’ ’ before MS before MS

SCX : strong cation exchange ; HILIC : hydrophobic interaction chromatography

6 Liquid samples: removal of Liquid samples: removal of ‘ ‘contaminants contaminants’ ’ before MS before MS

Better than all the above Better than all the above

Design protocols to avoid use of some of (these) contaminants Design protocols to avoid use of some of (these) contaminants not always possible but worth a try not always possible but worth a try

• Reduce detergents

Reduce detergents

• Substitute polymeric detergents (NP-40, Triton,TWEEN) with

Substitute polymeric detergents (NP-40, Triton,TWEEN) with

• thers with low molecular weight and defined molecular composition:
• thers with low molecular weight and defined molecular composition:

CHAPS, CHAPS, Octyl Octyl Glucoside Glucoside

• Use volatile buffers ( ammonium acetate, ammonium bicarbonate)

Use volatile buffers ( ammonium acetate, ammonium bicarbonate) in final steps in final steps

• Include desalting at the end of purification schemes

Include desalting at the end of purification schemes

Interfacing biochemistry to MS Interfacing biochemistry to MS

• SDS-PAGE : 1D (or 2D) are a nearly

universal interface between sample preparation and MS (for protein ID or PTM studies)

• LC-based approaches : more care is

required in planning upstream experiments

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• Ideal interface to biology
• Analytical and micropreparative
• Robust
• Solid phase chemistry of proteins
• Easy, low-tech
• Removal of contaminants :

– At the loading point – After migration during fix / staining steps

Why is SDS-PAGE such a good preparation method? Why is SDS-PAGE such a good preparation method?

• protein digestion in gel: non quantitative
• peptide sequence recovery: usually incomplete
• whole protein recovery: poor

Disadvantages

In-gel digestion: solid phase chemistry of proteins In-gel digestion: solid phase chemistry of proteins

8 From 1D- and 2D-PAGE to MS: sample preparation From 1D- and 2D-PAGE to MS: sample preparation

Protease digestion (Trypsin) Peptide extraction MS m/z Desalting on rp column

Sequence database

Best matching sequence

• Spot cutting and sample processing can be automated
• SDS-PAGE and reversed-phase LC are the main final

interfaces between biology and Mass Spectrometry; these techniques can be used both to clean up and separate proteins before MS

• Many compounds commonly used in protein purification

are incompatible with MS

• Hence: every new experiment should be planned from

the beginning to optimise the preparation of the sample for MS

– Efficiency – Sensitivity – Fractionation of complex mixtures

Interfacing to MS: conclusions Interfacing to MS: conclusions

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Introduction to MALDI and Electrospray mass spectrometry

Manfredo Quadroni www.unil.ch/paf

• > m/z (mono) of VATVSLPR 1+ : ( 841.502 + 1.008 ) /1 = 842.510
• > m/z (mono) of LGE…AAK 3+ : ( 2210.097 + (3 x 1.008 )) /3 = 737.707

Sequence Composition MW(mono) MW(ave) m/z (mono) m/z(ave)

• VATVSLPR

C37.H68.N11.O11 841.5022 841.999 842.510 843.014 LGEHNIDVLEGNEQFINAAK C96.H152.N27.O33 2210.097 2211.397 2211.105 2211.105

Isotope Atomic mass % Abundance Element Mr(mono.) Mr(avg.) 1H 1.0078 99.985 H 1.0078 1.0080 2H 2.0141 0.015

• 12C

12.0000 98.93 C 12.0000 12.0107 13C 13.0034 1.07

• 35Cl

34.9689 75.78 Cl 34.9689 35.4525 37Cl 24.22 24.22

m/z = (m + (mA * z )) / z Mass distribution and mass measurements

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839.0 840.6 842.2 843.8 845.4 847.0 Mass (m/z) 10 20 30 40 50 60 70 80 90 100 842.502 843.485 844.480 % Intensity 2209.0 2210.6 2212.2 2213.8 2215.4 2217.0 Mass (m/z) 10 20 30 40 50 60 70 80 90 100 2212.120 2213.137 2211.097 2214.149 % Intensity

Avg. Avg. Mono. Mono.

Singly charged ions as an example

850 848 846 844 842 100 90 80 70 60 50 40 30 20 10

Calculated with resolution 1000 Calculated with resolution 10000 Theoretical MS : the effect of resolution Theoretical distribution

11

Modes of measurement

• Ion production (ionisation)
• Ion separation
• Ion detection
• Ion production (ionisation)
• Ion separation – isolation of “parent” ion
• Ion fragmentation
• Ion detection - measure fragments of the “parent” ion

MS MS/MS

• MALDI :

– Solid phase (crystal) – Mostly single charged ions (for tryptic peptides)

• ESI :
• liquid phase
• mostly 2+ or 3+ ions (for tryptic

peptides)

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MALDI (Matrix Assisted Laser Desoprtion Ionisation MALDI TOF (Time Of Flight)

+TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS ... a=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded

• Max. 1305.0 counts.

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z, amu 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 847.59 1440.00 1479.98 1567.94 1640.16 1022.56 869.07 1305.87 1163.77 1481.98 1296.86 1249.77 1050.55 871.07 1024.56 1417.93 1283.91 789.53 1142.86 1443.01 1073.03 1595.95 1386.76 1824.09 857.14 1108.71 1292.95 978.60 1790.10 1501.84 1616.92

MALDI-TOF of a tryptic digest of BSA

YLYEIAR LSQKFPK LVNELTEFAK FKDLGEEHFK HPEYAVSVLLR HLVDEPQNLIK LGEYGFQNALIVR KVPQVSTPTLVEVSR DAFLGSFLYEYSR

? ? ? ? ? ?

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MALDI-TOF

• Great for Peptide Mass Fingerprinting

– Fast – Easy to measure – Sensitive – Salt-tolerant (to some extent) – Also good for larger MW (small proteins) – Sample on a stable support (no time constraints) – 1+ ions  simpler data analysis

• High accuracy needs careful calibration
• Difficult to do MS/MS by MALDI
• Signal suppression in complex mixtures

disadvantages

+ + + + + + + + + +

• -
• +

+

• +
• +

+

• +

+

• +
• +

+

• +

+ + ++ + + + + + + + + + + + HV power supply e- e- Taylor cone detector Front plate with orifice + + + + + + + High vacuum Region

Mechanism of ElectroSpray Ionisation

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ESI : rule of thumb Signal intensity ~ concentration

low flow rates give maximum sensitivity Trend towards capillary- and nano-HPLC

+TOF MS: Experiment 1, 57.059 to 57.093 min from BSA 1.wiff a=3.55486598098460260e-004, t0=7.07516527348161620e+001

• Max. 202.0 counts.

580.0580.5 581.0581.5582.0 582.5583.0583.5 584.0584.5585.0 585.5586.0 m/z, amu 20 40 60 80 100 120 140 160 180 200 Intensity, counts 582.28 582.77 583.27 +TOF MS: 43 MCA scans from Sample 2 (BSA Digest 100 fmol 5000) of BSA Digest 100 fm... a=3.56217430068478150e-004, t0=3.64725878201043440e+001

• Max. 282.0 counts.

1160 1162 1164 1166 1168 1170 1172 1174 m/z, amu 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 84 Intensity, counts 1163.76 1164.76 1165.74 1166.78 1162.70

Looking at 1+ vs. 2+ ions

Δm=1.0 Da 1.0 Da 1.0 Da Δm=0.5 Da 0.5 Da 0.5 Da

1+ 2+

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2+ and 3+ ions detected in the same scan

+TOF MS: Experiment 1, 26.119 to 26.788 min from w212_tissot_M23_12.wiff a=3.56223793209098900e-004, t0=7.12463273130560990e+001

• Max. 587.8 counts.

450 500 550 600 650 700 750 800 850 900 m/z, amu 50 100 150 200 250 300 350 400 450 500 550 588 486.94 738.38 792.85 421.77 741.34

3+ 2+ MH+=1458.7

ESI-MS

• Great for MS/MS

– Can be directly coupled to LC (separation !) – Sensitive – Excellent for MS/MS due to 2+/3+ ions

• Sample introduction more complex
• Data analysis more difficult (2+/3+ ions)
• one-shot sample analysis
• Less tolerant to contaminants

disadvantages

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Transmit Scan Select Transmit Fragment Transmit Scan Select Transmit Scan Select Transmit Fragment Transmit Trap Select Fragment Scan

Q1 Q2 Q3 Q1 Q2 Q3-LIT Q1 Q2 TOF

Transmit Scan Select Transmit Fragment Scan

3D - IT

Trap Select Fragment Scan

Triple Quadrupole Triple quadrupole – linear ion trap Quadrupole-Quadrupole- TOF 3D-ion trap (Paul trap)

Most common MS/MS-capable, ESI-based instruments

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REFLECTRON SKIMMER SAMPLING CONE ELECTROSPRAY NEEDLE HEXAPOLE GAS COLLISION CELL

DETECTOR PUSHERTOF

RF HEXAPOLE RF HEXAPOLE

Quadrupole Time of flight Mass Spectrometer (QTof)

electrospray source

• Two main strategies for protein ID :

– PMF with MALDI-TOF MS data – Db Search with MS/MS data (ESI or MALDI)

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M1 M2 M3 M4 M5 M6 M7 M8 M8 M7

...

Peptide Fragment Fingerprinting Search with multiple (20- 300 ) CID spectra Full scan of a mix of tryptic peptides M6

In-depth analysis by MS / MS of single peptides

CID

• n individual

peptides Qq-TOF

• Equilibrate pH
• Reduce -SH
• Alkylate w. I-Ac-NH2
• Dehydrate
• Rehydr. W Trypsin
• Digest 37 C
• Extract w. 30% MeCN, 5% formic acid
• Repeat extraction
• Poole extracts, reduce volume
• Concentrate 1/4, spot on MALDI plate
• Keep 3/4 for LC-MS if necessary

M1 M2 M3 M4 M5 M6 M7 M8

Peptide Mass Fingerprinting Search with mass list

Sample preparation and first-pass analysis by PMF

MALDI-TOF M1......M8

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• NANO-HPLC
• LC-PACKINGS ULTIMATE
• Inject 5 µl ; preconcentration on 300 µm ID x 1 mm C18 column at 20 µl / min
• 75 µm ID PepMap C18 column
• 120 min. gradient : 200 nl / min, 0-35 % MeCN in 105 min
• New Objective tip spraying at 2300 V
• SURVEY SCAN :
• Scan range 400-1200 Da, 1 s
• Enhance mode
• IDA CRITERIA :
• Pick two - three most intense peaks in the range 400-1200
• Charge state restricted to m/z=2,3,4
• Intensity threshold : 6 cps
• Exclude analysed masses for 300 seconds
• Exclude charge states within a 5 Da window
• Exclusion mass list : trypsin, keratin, myosin, a few ribosomal proteins
• DB SEARCH : MASCOT
• Tolerance 0.3 Da for peptide mass, 0.3 Da for fragment masses

LC-MS IDA parameters LC-MS IDA parameters

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+TOF Product (577.3

100 200 300 400 500 600 700 800 900 1000 1100 m/z, amu 100 200 300 400 500 600 Intensity, counts 245.12 316.16 577.31 838.54 181.13 252.16 147.14 288.17 169.13 545.32 457.30

+TOF MS: 18.026 to 18.135 min

400 500 600 700 800 900 1000 1100 1200

m/z, amu

200 400 600 800 1000 1200 Intensity, counts 577.30 594.27 568.31 588.31 446.77 706.46 605.27 1153.59 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

Time, min

1.0e4 5.0e4 1.0e5 1.3e5

Intensity, cps

245.12 737.40 175.10 588.89 421.77 901.54 442.75 586.26 787.88 489.21 547.55 409.18 120.11 409.17

1 2 3 4

LC-MS : data collection routine

• Full scan
• Detect peaks, make list
• Determine charge states
• immediate (QTOF, high resolution)
• zoom scan (ion traps)
• Choose ions with desired charge state (2+ - 4+)
• Exclude previously analysed ions
• Do MS/MS on X most intense peaks

Cycle time must be compatible with LC time scale (one peak ~ 30-50 s)  Usually 3 – 15 s

LC-MS : data collection routine

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C O C H R N H O C N H C H R H C H R N H C O OH

2 1 3

y2 b1

O C N H C H R H

1

+

C O C H R N H C H R N H C O OH

2 3

H

+

N H C H R H

1

+

C O C H R N H O C C H R N H C O OH

2 3

+ a1 z2

N H O C N H C H R H

1

H

+

C O C H R C H R N H C O OH

2 3

+ c1 x2

Peptide Fragmentation In the Gas phase

Product (653.3) 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z, amu 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 19.7 Intensity, counts 251.12 110.06 223.13 138.05 653.36 332.21 594.25 86.09 350.24 712.46 1055.49 373.30 566.32 465.16 956.41 1168.56 166.05 841.53 206.16 a1 b1 I a2 b2 y10 I b3 y9 b4 y8 a5 b5 y7 y6 b8 y3 I y2 y1

MH22+

precursor

Residue Immonium a b y

• H, His

110.07 110.07 138.06 1305.71 L, Leu 86.09 223.15 251.15 1168.65 V, Val 72.08 322.22 350.21 1055.57 D, Asp 88.03 437.25 465.24 956.50 E, Glu 102.05 566.29 594.28 841.47 P, Pro 70.06 663.34 691.34 712.43 Q, Gln 101.07 791.40 819.39 615.38 N, Asn 87.05 905.44 933.44 487.32 L, Leu 86.09 1018.53 1046.52 373.28 I, Ile 86.09 1131.6157 1159.61 260.19 K, Lys 101.10 1259.7106 1287.70 147.11

Table of theoretical fragments for a peptide sequence

22

Mascot search output Mascot search output

Mascot Search Results

Significant hits: ALBU_BOVIN (P02769) Serum albumin precursor (Allergen Bos d 6). ALBU_CANFA (P49822) Serum albumin precursor (Allergen Can f 3). VWF_PIG (Q28833) Von Willebrand factor precursor (vWF) (Fragment). CIQ3_BOVIN (P58126) Voltage-gated potassium channel protein KQT-like 3 RYR2_RABIT (P30957) Ryanodine receptor 2 (Cardiac muscle-type ryanodin K2CA_BOVIN (P04263) Keratin, type II cytoskeletal 68 kDa, component IA ALFB_RABIT (P79226) Fructose-bisphosphate aldolase B (EC 4.1.2.13) (Li

ALBU_BOVIN Mass: 71244 Total score: 711 Peptides matched: 12 (P02769) Serum albumin precursor (Allergen Bos d 6). Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide 15 461.80 921.58 921.48 0.10 0 55 1 AEFVEVTK 19 501.80 1001.58 1001.58 0.01 0 31 1 LVVSTQTALA 31 569.80 1137.58 1137.49 0.09 0 72 1 CCTESLVNR 33 582.30 1162.58 1162.62 -0.04 0 76 1 LVNELTEFAK 47 653.40 1304.78 1304.71 0.08 0 90 1 HLVDEPQNLIK 58 722.40 1442.78 1442.63 0.15 0 87 1 YICDNQDTISSK 60 739.80 1477.58 1477.52 0.07 0 43 1 ETYGDMADCCEK 61 740.40 1478.78 1478.79 -0.00 0 61 1 LGEYGFQNALIVR 64 751.90 1501.78 1501.61 0.18 0 37 1 EYEATLEECCAK 74 547.30 1638.88 1638.93 -0.05 1 85 1 KVPQVSTPTLVEVSR 97 627.70 1880.08 1879.91 0.16 0 34 1 RPCFSALTPDETYVPK 98 636.70 1907.08 1906.91 0.16 0 42 1 LFTFHADICTLPDTEK

• 1) Extract MS/MS spectra
• 2) Clean up : - noise subtraction
• smooth
• de-isotope
• centroid
• 3) Write text file : - precursor mass
• mass/intensity list

SEARCH=MIS REPTYPE=Peptide BEGIN IONS PEPMASS=440.8 CHARGE=2+ TITLE=Elution from: 23.72 to 23.77 173.1516 1.5 187.1531 1 219.1771 1.5 306.1851 1 323.2265 3 341.499 4 389.7626 1 410.8224 1 422.6573 3 431.8544 1 441.2406 95 443.2499 1 END IONS Next spectrum ….. …

Data export

!

Loss of information possible

23 Orthogonal datasets and confidence levels

Db : 100’000 sequences Probability of one (any) spectrum “accidentally” matching a sequence (wrong match) : 1/100’000 x 500 = 5.10-3 (0.005) Probability of 2 spectra “accidentally” matching the same sequence (wrong match) : 5.10-3 x 5.10-3 = 2.5 x 10-5 500 spectra Much higher confidence of identification with at least 2 peptides matching the same protein sequence

++ ++ usually 2D-LC is needed

• MudPit

+++ ++ usually 2D-LC is needed (+)

• ICAT

+* ++ ++

• De novo

Sequencing ++ ++ (+) (+)

• PTM analysis

Pure protein +++ ++ (+)

• Protein id

(complex mixture) +++ +++ ++ (+) (+) Protein id (1D gel band) +++* +++* +++ +++ ++ Protein id (2D gel spots) MS/MS MS/MS MS/MS PSD PMF Database searching Input LC-MALDI- TOF-TOF (MS/MS) x LC-ESI-IT LC-ESI-QTOF MALDI-TOF- TOF (MS/MS) MALDI-TOF (PSD) MALDI-TOF * means that this technique is even excessively good for the task

x we mean here the LC separation of peptides in-line with deposition of fractions on the MALDI plate and

subsequent extensive peptide analysis by MALDI-TOF-TOF

HOW MUCH MASS SPECTROMETRY IS NEEDED ? HOW MUCH MASS SPECTROMETRY IS NEEDED ?

24

WORKFLOWS 1: WORKFLOWS 1: classical classical 2D-PAGE 2D-PAGE + + MALDI TOF MALDI TOF

1 1 2 2 3A 3A 3B 3B 4 4 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 1 14 8 15

Workflow 1: adaptation of bacteria to growth conditions Workflow 1: adaptation of bacteria to growth conditions

Normal medium Low Glucose

Wick LM, et al, Environ Microbiol 3: 588-599, 2001

E.Coli adapts to a very low glucose medium by up- and downregulating a set of 15 proteins

25

M1 M2 M3 M4 M5 M6 M7 M8 Peptide Mass Fingerprinting Search with mass list

M1......M8

Tryptic digestion

amino acid transport argT P09551 5.22 25.8 12 sugar transport rbsB P02925 7.76 30.9 11 catabolism gatY P37192 5.81 61.5 10 sugar transport mglB P02927 5..25 33.36 9 sugar transport malE P02928 5.22 40.7 8 amino acid transport livJ P02917 7.03 41.3 7 transport ydcS P76108 ? ? 6 proton transport/energy atpA P00822 6.02 55.2 5 transport ugpB P10904 6.71 48.45 4 Central metabolism aceA P05313 5.06 47.5 3 peptide transport dppA P23847 6.21 60.3 2 central metabolism aldA P25553 5.07 52.2 1 Function Name

• Acc. N.

pI* mw* Nr

Workflow 1: adaptation of bacteria to growth conditions Workflow 1: adaptation of bacteria to growth conditions

Metabolic enzymes and transport proteins affected Utilisation of alternative sources of energy

Workflow 1: quantitation and kinetics Workflow 1: quantitation and kinetics

UgpB (4) 0.02 0.04 0.06 0.08 0.10 0.12 1 2 3 4 5 DppA (2) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 1 2 3 4 5 AldA (1) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1 2 3 4 5 AceA (3A) 1 2 3 4 5 6 1 2 3 4 5 AceA (3B) 0.2 0.4 0.6 0.8 1.0 1.2 1 2 3 4 5 AtpA (5) 0.05 0.10 0.15 0.20 0.25 0.30 0.35 1 2 3 4 5 YdcS (6) 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 1 2 3 4 5 LivJ (7) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 1 2 3 4 5 MalE (8) 2 4 6 8 10 12 14 1 2 3 4 5 GatY (10) 0.5 1.0 1.5 2.0 2.5 3.0 1 2 3 4 5 RbsB (11) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2 3 4 5 MglB (9) 2 4 6 8 10 12 14 16 18 1 2 3 4 5 ArgT (12) 0.5 1.0 1.5 2.0 2.5 3.0 1 2 3 4 5 MalI (13) 0.05 0.10 0.15 0.20 0.25 1 2 3 4 5

Relative protein quantities as a function of time during the adaptation process

Time points : 1) Start – batch culture 2) Adapted bacteria put back into high- glucose batch culture 3) 40 hr adaptation culture 4) 156 hr adaptation culture 5) 500 hr adaptation culture

26

WORKFLOWS 2: WORKFLOWS 2: Affinity pull-down Affinity pull-down + + 1D-PAGE 1D-PAGE + + LC-MS/MS LC-MS/MS

TNF family of ligands and TNF-receptor family TNF family of ligands and TNF-receptor family

Bodmer JL et al, TIBS. 2002 27(1):19-26

27

(-) FasL (+) FasL

FADD

Casp.8 Fas

Analysis of apoptotic signalling complexes Analysis of apoptotic signalling complexes

Real life Model

The Fas (CD95) signalling complex (DISC)

Flip Casp.10 Fc-FasL

? ? Western blot

(-) BAFF (+) FasL

Analysis of apoptotic signalling complexes: negative control Analysis of apoptotic signalling complexes: negative control

* run 1 cm * no fix, no stain ! * cut, digest

1 cm

(-) BAFF (+) FasL

28

LCMS runs Fas/Baff

TIC: from 180702BaffHL.wiff

• Max. 3.8e5 cps.

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 Time, min 0.0 2.0e4 4.0e4 6.0e4 8.0e4 1.0e5 1.2e5 1.4e5 1.6e5 1.8e5 2.0e5 2.2e5 2.4e5 2.6e5 2.8e5 3.0e5 3.2e5 3.4e5 3.6e5 3.8e5 TIC: from FAS_180702_HL.wiff

• Max. 4.0e5 cps.

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 Time, min 0.0 2.0e4 4.0e4 6.0e4 8.0e4 1.0e5 1.2e5 1.4e5 1.6e5 1.8e5 2.0e5 2.2e5 2.4e5 2.6e5 2.8e5 3.0e5 3.2e5 3.4e5 3.6e5 3.8e5 4.0e5 603.33 655.82 328.19 130.11 405.33 337.17 864.45 171.17 582.29 643.87 603.69 158.06 186.16 171.18 607.38 120.11 620.67 449.15

BAFF complex HL sample FasL complex HL sample 600 spectra 680 spectra

LC-MS/MS ANALYSIS LC-MS/MS ANALYSIS

BAFF HL search results

Database : MSDB 200402 (851746 sequences; 265326103 residues) Taxonomy : Mammalia (mammals) (166849 sequences)

Significant hits:

Q96RF8 SSA1.- Homo sapiens (Human). BAB27292 AK010960 NID: - Mus musculus K2C1_HUMAN Keratin, type II cytoskeletal 1 (Cytokeratin 1) A45935 dnaK-type molecular chaperone hsc70 - mouse KRHU0 keratin 10, type I, cytoskeletal - human C25437 tubulin beta-3 chain - mouse Q9CWA2 ATP SYNTHASE, H+ TRANSPORTING MITOCHONDRIAL F1 CAA30026 HSHA44G NID: - Homo sapiens CAB59134 SEQUENCE 1 FROM PATENT WO9818921 PRECURSOR A44861 keratin, 67K type II epidermal - human Q9BWB7 HEAT SHOCK 70KD PROTEIN 9B (MORTALIN-) (Human). A26168 ribophorin I precursor - human PT0207 Ig gamma chain C region - chimpanzee PWHUA H+-transporting two-sector ATPase alpha chain precursor - human JC1473 H+-transporting ATP synthase (EC 3.6.1.34) alpha chain - mouse I77403 tubulin alpha-1 chain - human AAA57233 MUSHP7A2 NID: - Mus musculus CAA82315 HSKERAT9 NID: - Homo sapiens A29904 keratin 5, type II, epidermal - human HHHU84 heat shock protein 90-beta [validated] - human AAB86467 IMMUNOGLOBULIN GAMMA HEAVY CHAIN .-(Human). B26168 ribophorin II precursor - human CAA34756 HSEF1AC NID: - Homo sapiens S21097 alpha-1-antitrypsin precursor - bovine S04652 Ca2+-transporting ATPase (EC 3.6.1.38) 2, - pig CAA41735 BTBSA NID: - Bos taurus Q9UK02 BIP PROTEIN (FRAGMENT).- Homo sapiens (Human). KRHUEA keratin 6a, type II - human A22224 actin alpha, vascular smooth muscle - mouse Q96GA6 UNKNOWN (PROTEIN FOR MGC:15420).- Homo sapiens (Human). Q8WZ42 TITIN.- Homo sapiens (Human). I84741 RNA helicase - mouse Q9TS10 78 KDA APAMIN BINDING PROTEIN.- Bos taurus (Bovine). AAH02690 BC002690 NID: - Homo sapiens I48385 RNA helicase TNZ2 - mouse Q96FZ6 HEAT SHOCK 60KD PROTEIN 1 (CHAPERONIN).(Human). AAA56753 HSU15637 NID: - Homo sapiens CAA58470 HSPXMP11 NID: - Homo sapiens JQ0028 cytokeratin 19 – mouse …………………………………

FasL HL search results

Database : MSDB 200402 (851746 sequences; 265326103 residues) Taxonomy : Mammalia (mammals) (166849 sequences)

Significant hits:

A37241 52K autoantigen Ro/SS-A - human Q8WUC1 TUBULIN, BETA 5.- Homo sapiens (Human). C25437 tubulin beta-3 chain - mouse CAC39526 SEQUENCE 15 FROM PATENT WO0129232.-HUman AAH19046 SIMILAR TO IMMUNOGLOBULIN HEAVY CONSTANT GAMMA 3 K1CJ_HUMAN Keratin, type I cytoskeletal 10 (Cytokeratin 10-(Human). I37383 FAS soluble protein - human A24903 tubulin alpha-1 chain - Chinese hamster A44861 keratin, 67K type II epidermal - human I38707 Fas ligand - human AAG41947 AF304164 NID: - Homo sapiens Q9BDN1 CD95L PROTEIN.- Cercocebus torquatus atys CAA82315 HSKERAT9 NID: - Homo sapiens AAB86467 IMMUNOGLOBULIN GAMMA HEAVY CHAIN (Human). NUCL_HUMAN Nucleolin (Protein C23).- (Human). 1ATS heat shock cognate protein 70 kD (44 kD chaperone ATPase 1FC1A Ig gamma-1 chain C region (Fc fragment), chain A - human I61769 keratin 6d, type II - human (fragment) A29904 keratin 5, type II, epidermal - human A40389 translation elongation factor eEF-1 alpha chain (clone pS1) - rat AAB46730 HSU86214 NID: - Homo sapiens CAD23746 IMMUNOGLOBULIN GAMMA HEAVY CHAIN CONSTANT REGION Q10466 TITIN, HEART ISOFORM N2-B (EC 2.7.1.-) (CONNECTIN).-(Human). Q8WZ42 TITIN.- Homo sapiens (Human). 1D3OA trypsin (EC 3.4.21.4), chain A - pig CAC20457 IMMUNOGLOBULIN HEAVY CHAIN CONSTANT GAMMA 4.- (Human). O75634 HEAT SHOCK PROTEIN 70 TESTIS VARIANT.- (Human). Q8WTZ6 RIBOSOMAL PROTEIN L18.- Homo sapiens (Human). 1NBMC f1-atpase (EC 3.6.1.34) delta and 1 epsilon subunits, chain C - bovine Q96PE2 TUMOR ENDOTHELIAL MARKER 4.- Homo sapiens (Human). Q9R1Q3 GLIAL FIBRILLARY ACIDIC PROTEIN ALPHA.- Rattus norvegicus (Rat). ITSH alpha-1-antitrypsin precursor - sheep CAA27396 MMACTBR2 NID: - Mus musculus Q8WXH0 NUANCE.- Homo sapiens (Human). Q9GL40 FAS ANTIGEN.- Macaca mulatta (Rhesus macaque). A33370 H+-transporting ATP synthase beta chain precursor, mitochondrial CAB76567 MMU250841 NID: - Mus musculus Q9GK28 FAS ANTIGEN APO-1/CD95.- Macaca arctoides (Stump-tailed macaque). Q9BZL4 MYOSIN BINDING SUBUNIT 85.- Homo sapiens (Human). ………………………………… Database search 1. Protein 1 2. Protein 2 3. Protein 3 4. … Database search Database search 1. Protein 1 2. Protein 2 3. Protein 3 4. … 1. Protein 1 2. Protein 2 3. Protein 3 4. … 1. Protein 1 2. Protein 2 3. Protein 3 4. Protein 4 5. Protein 5 6. Protein 6 7. …

Total list sample 1

1. Protein 1 2. Protein 2 3. Protein 3 4. Protein 4 5. Protein 5 6. Protein 6 7. …

Total list sample 2

1. Protein 1 2. Protein 2 3. Protein 3 4. Protein 4 5. Protein 5 6. Protein 6 7. …

Total list sample 1

1) COMMON CONTAMINANTS ( PROTEINS STICKING TO BEADS) 2) LIGAND-COPURIFYING PROTEINS (EX SJOGREN SYNDROME 52 KDA) 3) COMMON HITS (WHAT IS IN BOTH LISTS)

Subtract from each list : FILTERED RESULTS FILTERED RESULTS

29 FILTERED RESULTS FILTERED RESULTS

EF11_HUMAN (P04720) Elongation factor 1-alpha 1 (EF-1-alpha-1) (Elonga EF11_HUMAN (P04720) Elongation factor 1-alpha 1 (EF-1-alpha-1) (Elonga T13B_HUMAN (Q9Y275) Tumor necrosis factor ligand superfamily member 13 (BAFF) RS3A_MOUSE (P97351) 40S ribosomal protein S3A RS3A_MOUSE (P97351) 40S ribosomal protein S3A CLUS_HUMAN (P10909) Clusterin precursor CLUS_HUMAN (P10909) Clusterin precursor K22E_HUMAN (P35908) Keratin K22E_HUMAN (P35908) Keratin PHB_HUMAN (P35232) Prohibitin ICEA_HUMAN (Q92851) Caspase-10 RL7_HUMAN (P18124) 60S ribosomal protein L7 RL7_HUMAN (P18124) 60S ribosomal protein L7 RL7A_MOUSE (P12970) 60S ribosomal protein L7a RL7A_MOUSE (P12970) 60S ribosomal protein L7a TNR6_HUMAN (P25445) TNF-family receptor CD95 GBLP_HUMAN (P25388) Guanine nucleotide-binding protein GBLP_HUMAN (P25388) Guanine nucleotide-binding protein RS8_HUMAN (P09058) 40S ribosomal protein S8 CFLA_HUMAN (O15519) CASP8 and FADD-like apoptosis regulator GC1_HUMAN (P01857) Ig gamma-1 chain C region GC1_HUMAN (P01857) Ig gamma-1 chain C region K2C1_HUMAN (P04264) Keratin K2C1_HUMAN (P04264) Keratin FADD_HUMAN (Q13158) FADD RS3_HUMAN (P23396) 40S ribosomal protein S3 RS3_HUMAN (P23396) 40S ribosomal protein S3 RO52_HUMAN (P19474) 52 kDa Ro protein (Sjogren syndrome type A antigen RO52_HUMAN (P19474) 52 kDa Ro protein (Sjogren syndrome type A antigen ICE8_HUMAN (Q14790) Caspase-8

MASCOT DATABASE SEARCH BAFF receptor complex MASCOT DATABASE SEARCH Fas (CD95) complex

SDS-PAGE as an interface biology SDS-PAGE as an interface biology   MS MS

Disadvantages

• In-gel digestion : low quantitative reproducibility
• > sample-to-sample comparisons difficult
• In-gel digestion : sequence coverage usually incomplete

Develop methods avoiding gel based steps

30

WORKFLOWS 3: WORKFLOWS 3: Analysis of a Analysis of a post-translational post-translational modification modification

TF65_HUMAN Mass: 60705 Total score: 523 Peptides matched: 23 (Q04206) Transcription factor Query Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide 10 416.84 831.68 831.50 0.18 0 32 1 QVAIVFR 27 449.53 897.06 896.48 0.57 0 13 1 DPSGRPLR 67 511.72 1021.44 1021.48 -0.05 0 17 1 GDYDLNAVR 84 530.18 1058.36 1058.54 -0.18 0 39 1 DLEQAISQR 85 530.20 1058.40 1058.54 -0.14 0 (35) 1 DLEQAISQR 120 567.69 1133.38 1133.58 -0.21 0 55 1 INGYTGPGTVR 121 567.92 1133.84 1133.58 0.25 0 (16) 1 INGYTGPGTVR 209 653.19 1304.38 1304.63 -0.26 1 41 1 TYETFKSIMK + Acetyl (K); Oxidation (M) 274 478.54 1432.61 1432.73 -0.11 2 (22) 1 TYETFKSIMKK + Acetyl (K); Oxidation (M) 296 738.20 1474.40 1474.74 -0.34 2 56 1 TYETFKSIMKK + 2 Acetyl (K); Oxidation (M) 297 492.49 1474.46 1474.74 -0.27 2 (29) 1 TYETFKSIMKK + 2 Acetyl (K); Oxidation (M) 316 503.14 1506.41 1506.76 -0.34 0 33 1 SPFSGPTDPRPPPR 318 756.22 1510.44 1510.78 -0.34 0 38 1 TPPYADPSLQAPVR 360 539.15 1614.44 1614.84 -0.39 0 13 3 DPPHRPHPHELVGK 361 404.64 1614.55 1614.84 -0.29 0 (6) 2 DPPHRPHPHELVGK 369 545.81 1634.42 1634.85 -0.43 1 (23) 1 KSPFSGPTDPRPPPR 385 555.20 1662.59 1662.86 -0.27 1 38 1 SPFSGPTDPRPPPRR 387 416.85 1663.39 1662.86 0.53 1 (16) 1 SPFSGPTDPRPPPRR 395 559.87 1676.60 1676.86 -0.26 1 25 1 KSPFSGPTDPRPPPR + Acetyl (K) 411 568.50 1702.49 1702.83 -0.34 0 34 1 CIHSFQNLGIQCVK 473 612.18 1833.53 1832.96 0.57 2 23 1 KSPFSGPTDPRPPPRR + Acetyl (K) 514 963.79 1925.58 1925.96 -0.38 0 75 1 IQTNNNPFQVPIEEQR 515 642.88 1925.63 1925.96 -0.33 0 (37) 1 IQTNNNPFQVPIEEQR

Q04206 is modified by a nuclear acetyltransferase

• Max. 4.0e5 cps.

Trypsin digest

Mass tol. 0.8, 3 missed cleavages, acetyl(K) as variable modification

31

TOF PreCursor 0 MCA scans from 161203_QS_MQ_Ac_p65_par126.wiff Precursor of 12...

• Max. 86.0 counts.

400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 m/z, amu 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Intensity, counts

502.10 644.43 755.56 559.11 529.40 682.66 613.02 729.74 545.02

695.09

477.12

587.22 627.57

+TOF MS: Experiment 1, 7.182 to 9.771 min from 161203_QS_MQ_15min_p65_Ac.wiff a=3.56275471721098790e-004, t0=7.24150134716619500e+001

• Max. 74.7 counts.

400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 m/z, amu 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Intensity, counts 530.21 502.93 756.37 567.74 504.94 480.97 736.28 613.74 482.96 643.27 637.59 468.55 539.23 672.36 506.94 464.55 539.56 654.28 605.32 687.34 460.23 752.90 743.36 559.22 472.23 497.99 645.27 771.38 702.35 673.36 613.28 564.29 571.25 453.43 708.62 633.26 531.72 470.19

Specific scan modes can be used to detect protein modifications

Tryptic digest Full scan Tryptic digest Par 126 scan

m/z= 126 is immonium ion -18