Mass spectrometry based proteomics

Mass

 

spectrometry

 

based

 

proteomics

Zsuzsanna Darula Institute of Biochemistry

November 25, 2015

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”

Proteomics

: large‐scale study of proteins

Proteome

: ‐the complete set of proteins

‐expressed by a cell / tissue / organism ‐at a given time

Separation

 

/

 

fractionation

 

of

 

protein

 

samples

 

1.:

 

gel

 

electrophoresis

1D: SDS‐PAGE (size separation, limited resolution, low‐complexity samples)

2D: IEF + SDS‐PAGE

isoelectric point + size

resolving power: a few thousand proteins/gel

limitations:  membrane proteins

highly acidic / basic proteins

very small/ large proteins

16‐BAC/SDS – PAGE

size‐separation in both dimensions, limited resolution

“Native gel” + SDS‐PAGE

analysis of protein complexes: isolation of complex in 1st _dimension, 

Type Abbr. Principle of separation

Size exclusion chromatography SEC Differences in size and shape

Ion exchange chromatography IEC Electrostatic interactions (pK_a, pK_b) Normal phase chromatography NPC

Polar interactions Hydrophilic interaction chromatography HILIC

Reversed phase chromatography RPC

Dispersive interactions Hydrophobic interaction chromatography HIC

Affinity chromatography AC Specific interactions

Separation

 

/

 

fractionation

 

of

 

protein

 

Identification

 

of

 

separated

 

proteins

 

1.

Edman

 

sequencing

→requires pure protein

→requires free protein N‐terminus (acetylation?) →~30 (max 50‐60) AA can be determined

→sample requirements: ≥10 pmol →slow (~ 1h / AA)

→ ~ isobaricAA’s can be distinguished (I/L; Q/K)

Amino Acid 1 AA 2 AA 3 AA 4 AA 5 REAGENT + Amino Acid 1 AA 2 AA 3 AA 4 AA 5 REAGENT Amino Acid 1 AA 2 AA 3 AA 4 AA 5 REAG ENT + H₂N H₂N HPLC  O HN H₂N R pH:8 H+_{, heat} H2N Peptide N NH S R O Phenylthiohydantoin

amino acid‐reagent product

N C S Peptide reagent first amino acid unit + + Phenylisothiocyanate (PITC)

→sensitive →specific →(semi)quantitative →requires antibody →foreknowledge of protein of interest is required →expensive

Identification

 

of

 

separated

 

proteins

 

2.

Identification

 

of

 

separated

 

proteins

 

3.

Mass

 

Spectrometry

 

(MS)

→ Sensitive (fmol/amol range) → Quick → No antibody or external standards required → Amenable with mixtures blocked peptides modified peptides (PTM analysis)

Mass

 

Spectrometry

 

(MS)

Determination of m/z value of gas‐phase ions  (mass‐to‐charge ration, m: mass, z: charge)

signal

ION SOURCE MASS ANALYZER DETECTOR

sample VACUUM SYSTEM Int. m/z 400 600 800 1000 spectrum

Ion

 

source

generation of gas‐phase ions

“soft” ionization techniques

MALDI: matrix assisted laser desorption ionization, singly charged ions

ESI: electrospray ionization, multiply charged ions

Mass

 

analyzer

separation of ions according to m/z

defines performance of the mass spectrometer

Sensitivity Resolution

Mass accuracy (absolute / relative (ppm!!!))

Linear dynamic range

Speed

Mass range

analyzers used in proteomics: Quadrupole, Ion trap, flight tube (TOF), FT‐ICR, 

R=500 R=1000

R=2000 R=10000

Element Mass  number Natural  occurrence  % C 12₁₃ 99₁ H 1₂ 99.99_0.01 O 16 17 18 99.76 0.04 0.2 N 14₁₅ 99.6_0.4 S 32 33 34 36 94.93 0.76 4.29 0.02 3000 2500 2000 1500 1000 500 0 1571 1570 1569 1568 1567 1567.69 Monoisotopic mass only 12_C, 1_H, 14_N, 16_O (and 32_S) 1 x 13_{C (and …)} 2 x 13_{C (and …)}

Monoisotopic peak is ALWAYS the first in the isotope cluster 

but not necessarily the most abundant!

m/z: 300 m/z: 1800

m/z: 5000 m/z: 3000

Δ m/z: 1 z=1 Δ m/z: 1/2 z=2 Δ m/z: 1/3 z=1

Isotope spacing enables charge determination

1 4 0 8 1 2 _ 4 8 _ 6 P M #1 9 0 7 R T : 1 3 .7 3 A V : 1 N L : 1 .6 7 E 5 T : F T M S + p N S I F u ll m s [3 8 0 .0 0 - 1 4 0 0 .0 0 ] 5 1 9 5 2 0 5 2 1 5 2 2 5 2 3 5 2 4 5 2 5 5 2 6 5 2 7 5 2 8 5 2 9 5 3 0 5 3 1 5 3 2 m /z 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 R e la tive A b u ndance 5 2 8 .9 2 6 5 2 9 .2 6 1 5 2 3 .2 8 5 5 1 9 .1 3 9 5 2 9 .5 9 5 5 2 3 .7 8 8 5 2 0 .1 3 9 5 2 8 .3 0 3 5 2 1 .1 3 6 5 2 9 .9 2 9 5 2 4 .2 8 9 5 2 2 .1 3 6 5 3 0 .2 6 4

MS

 

analysis

 

of

 

proteins

Top down Bottom up

Intact proteins are investigated Peptides generated from proteins 

are investigated

Peptide mass fingerprinting Tandem mass spectrometry

TOP

‐

DOWN

 

APPROACH

• molecular weight determination of intact proteins • fragmentation of intact isolated proteins (ETD, ECD, CID, HCD) degradation products sequence variants combinations of post‐translational modifications positions of disulfide bridges • techniqually challenging lower MW proteins limited sample complexity high resolution (R>200 000!), special instrument required

Bottom

 

up

 

approach

Generation of peptides from proteins

Enzymatically

Chemically

Cyanogen bromide (Met ↓  (Trp ↓)) Acidic hydrolysis (Asp↓, (Glu↓))

Endopeptidase Specificity pH‐range

Trypsin _R, K ↓ 7.5‐9.0 Chymotrypsin _{Y, F, W, L ↓} 7.0‐9.0 Glu C E, D ↓ 7.5‐8.5 Asp N _↓D 6.0‐8.0 Arg C _R ↓ 7.5‐8.5 Lys C _K ↓ 7.5‐8.5 Lys N _↓N 8.5‐9.5

Enzymatic

 

protocols

 

most

 

often

 

use

 

trypsin

• Cleaves after Lys and Arg (except the next AA is a Pro) • Provides at least one basic amino acid per peptide (facilitates ion generation for MS) • Statistically the size of the tryptic peptides are  perfect for m/z range of analyzers (~10% of  the  AA content is R or K) • Cheap, reliable, known sequence  • Modified trypsin is available commercially  (↓autolysis)

Peptide

 

mass

 

fingerprinting

 

(PMF)

2D gel 

electrophoresis reduction _trypsin

alkylation _extractionpeptide desalting

MALDI‐TOF Database

Search Int. m/z Protein list 1. 2. 3. MS compatible staining

Reduction: DTT (dithiothreitol), mercaptoethanol, TCEP (tris(2‐carboxyethyl)phosphine)

MALDI Ion Source (Matrix Assisted Laser Desorption Ionization)   H3CO HO OCH3 OH O HO OH O OH HO OH O CN

Commonly used matrices

DHB CHCA SA

2,5‐DihydroxyBenzoic Acid       α‐Cyano‐4‐HydroxyCinnamic Acid      Sinapinic Acid      

Laser beam

Protein or peptide analyte Acidic matrix molecule

Ta rg et   Pla te Desorption Desolvation + -+ + + -Ionization + + to Analyzer hν Co‐crystallized matrix 

and analyte molecules

TOF

 

Analyzer

(time

‐

of

‐

flight)

Repeller _Detector Plate Grid ( ‐pole) + + + +

Analytes with different m/z values

(different velocity → different amount of time to reach the detector)

Spectrum m/z Int. + Laser beam Signal + + +

generated ions are accelerated by applied electric field ions of same charge have the same kinetic energy

velocity of the ions depends on their mass‐to‐charge ratio time to reach the detector is measured, m/z calculated

2 2 2 L V t z m  or t  m z

9 0 0 1 1 0 0 1 3 0 0 1 5 0 0 1 7 0 0 1 9 0 0 2 1 0 0 m /z 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 1 2 0 0 0 1 4 0 0 0 a .i.

100

 

%

 

sequence

 

coverage?

‐ peptide m/z out of detection range (short and long peptides) ‐ poor recovery of hydrophobic peptides (extraction from gel) ‐ loss of hydrophilic peptides (desalting) ‐ ion suppression (MS analysis) ‐ incorrect sequence in database  ‐ protein processing (signal peptide, propeptide)

T

T

MALDI-TOF MS, without fractionation

PMF – SUMMARY

Protein is cleaved into peptides in a specific manner (usually 

using trypsin)

The m/z value of the resulting peptides is determined using 

mass spectrometry (typically MALDI‐TOF)

Protein is identified by database search by comparing 

experimentally determined peptide m/z’s to theoretical ones  

generated in silico from proteins present in database

For

 

more

 

complex

 

peptide

 

mixtures:

MS/MS

 

based

 

protein

 

ID

 

Tandem in space (two separate analyzers, Q‐TOF, Q‐TRAP, IT‐Orbitrap…)

Tandem in time (single analyzer, ion traps)

Int. m/z MS/MS (fragmentation)  spectrum sample + + + + ionization Ion source + -+ + ionization Analyzer 1

ion selection fragmentationionization

Detector

detection m/z separation

Multiply charged ions are formed On‐line coupling to HPLC: LC‐MS/MS + + + - ₊ + + + + + -(heated) capillary + + + + ++ ++ + + + + + + +++ ++ + + + ++ + + + + + + + + + + + + + + ++ + + + to the analyzer e -High voltage +

-Electrospray

 

ionization

 

(ESI)

Sample nebulized to fine aerosol

Size of sample droplets is reduced by applied electric field and heat (desolvation) Droplets explode when electrostatic repulsion overcomes surface tension

FRAGMENTATION

Energy‐based

Energy is put into the peptide (weakest bonds break)

Collision‐Induced Dissociation (CID/CAD, HCD)

Infra‐Red MultiPhoton Dissociation (IRMPD)

Radical‐based

Electrons create unstable radical ions that spontaneously fragment at 

sites of electron capture

Electron Capture Dissociation (ECD)

H₂N N H H N N H OH R₁ O R₂ O R₃ O R₄ O a₁ x₃ a₂ x₂ a₃ x₁ c₁ z₃ c₂ z₂ c₃ z₁ b₁ y₃ b₂ y₂ b₃ y₁ Sequence ions are formed by fragmentation of the peptide backbone:

y₁ y₂ y₃ y₄ y₅ y₆ S A M P L E R +_H 2 OH

b

fragment ions

y

fragment ions

S A M P L E R H +_H OH 2 + S A M P L R H +_H OH 2 + _E S A M P L H + +_H R OH 2 E S A M H + +_H L R OH 2 P E S A H S H + + L R OH +_H 2 P E L R OH +_H 2 A M P E M b₆ b₁ b₂ b₃ b₄ b₅ 6 2 1 3 4 5 6 5 4 3 2 1

Collision

 

induced

 

dissociation

 

(CID)

Instrument

‐

dependent

 

fragmentation

Fragment

 

ions

 

generated

 

by

 

CID:

1.

 

Sequence

 

ions (a,

 

b,

 

y)

2.

 

Internal

 

fragments

 

(if

 

multiple

 

fragmentation)

3.

 

Satellite

 

ions

 

(water

 

and

 

NH

₃

loss

 

of

 

fragment

 

ions)

4.

 

Immonium

 

ions

 

(info

 

on

 

amino

 

acid

 

content)

RT:0.00 - 89.88 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Time (min) 0 20 40 60 80 100 R e la tiv e A b u ndan ce 32.38 633.2811 63.80 711.1080 52.48 738.0400 38.21 704.3377 _40.64 531.8317 56.59 511.2694 47.84 535.2967 22.64 483.2597 31.21 _651.861368.43 421.7585 60.53 836.4495 _74.79 1066.5383 29.05 457.7869 83.26 942.1589 21.10 527.7853 2.27 445.1201 8.67 445.1200 11.87 445.1201 NL: 2.72E7 Base Peak F: ms MS 140325_27 140325_27 #8826 RT:41.43 AV:1 NL:4.20E6 T:FTMS + p NSI Full ms [380.00-1600.00] 400 450 500 550 600 650 700 750 800 850 900 950 m/z 0 20 40 60 80 100 R e la tiv e A b u ndan ce 521.3063 z=2 626.8540 z=2 672.8633 z=2 543.2941 z=2 477.7903 z=2 575.9614 z=3 441.2477 z=2 737.3463 z=2 801.4092_z=2 689.6523 z=3 486.7901 z=2 863.4384 z=2 602.3219 z=2 427.5571 z=3 654.3456_z=2 767.8786 900.9740_z=2 z=2 946.5130z=1 977.9644z=2 140325_27 #8827 RT:41.44 AV:1 NL:2.90E5

T:ITMS + c NSI d w Full ms2 [email protected] [160.00-1265.00]

200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 R e la tiv e A b unda nc e 742.4 671.3 557.3 855.5 743.4 582.3 511.3 398.3 672.4 1040.5 926.5 327.2 469.2 583.3 720.3 299.1 232.1 347.1 399.2 662.4 744.5 810.5 906.5 1021.6 213.1 952.7 1106.5 1164.8 1248.7 BPI MS MS / MS

Ions eluting from HPLC

Ions detected in  Orbitrap analyzer at 41.43min MS/MS spectrum of  precursor ion m/z:  626.854 (2+), fragmented and 

measured in ion trap

1. Generation of peak‐list (txt, mgf, dta …):

List of all MS/MS data acquired during an LC‐MS/MS experiment: For each MS/MS spectra:

precursor m/z, precursor charge state

list of m/z and intensity values for observed fragment ions 2. Database search

All MS/MS spectra is searched individually, results are given as proteins listing peptides 

assigned to them

Search engine (Mascot, Protein Prospector, OMSSA, Sequest, Proteome Discoverer, Byonic…)

1. defines peptide candidates with the same (theoretical) m/z that is observed for the 

precursor (in‐silico digestion of proteins in the database)

2. compares observed fragment ion list to theoretical fragment ion list of peptide 

candidates (instrument‐dependent fragmentation!!!)

3. assigns a score and / or a probability value (expectation value, E‐value) to peptide 

matches for deciding the “goodness” of identifications

One

 

peptide

 

to

 

multiple

 

proteins?

•

Homology

Between species Protein families within the same species Different isoforms of the same protein

•

Coincidence

Sample Gene Acc.No. Protein Protein MW

Unique Peptides

Coverage

1 At3g02090 Q42290 mitochondrial-processing peptidase beta subunit, (MPP beta)

59 32 55.00%

1 At3g16480 O04308 mitochondrial-processing peptidase alpha subunit 2, (MPP alpha-2)

54 1a _3.00%

1 At2g07727 P42792 Cytochrome b (MTCYB) (COB) (CYTB)

44.5 1b _3.30%

2 At1g51980 Q9ZU25 mitochondrial-processing peptidase alpha subunit 1 (MPP Alpha-1)

54c _{24 40.40%}

2 At3g16480 O04308 mitochondrial-processing peptidase alpha subunit 2, (MPP alpha-2)

54c ₁c_{(9) 16.40%}

3 At5g40810 Q0WNJ4 Cytochrome c1 (CYC1-2) 33 6 20.80% 4 At5g13430 Q9LYR3 Ubiquinol--cytochrome-c reductase

(REISKE subunit)

26 6 22.10%

5 At4g32470 Q9SUU5 Ubiquinol-cytochrome C reductase complex 14 kDa protein

14.5d _{5 49.20%}

5 At5g25450 Q3E953 Ubiquinol-cytochrome C reductase complex 14 kDa protein

All

 

MS/MS

 

data

 

assigned

 

to

 

peptides?

 

– NO

‐non‐peptide components (salts, detergents, derivatizing agents…)

‐incomplete reduction / alkylation upon sample preparation

‐post‐translational modifications

‐side reactions during sample preparation 

cyclization (N‐terminal Gln, Cys(CAM))

methylation (Glu, Asp)

oxidation (Met, Trp, Cys, Tyr)

carbamylation (N‐term, Lys)

carbamidomethylation (N‐term, Lys, Met, His, Asp)

deamidation (Asn, Gln)

S‐acrylamide formation (Cys)

formation of alkali metal adducts of peptides

‐nonspecific cleavages

‐incomplete digestion (number of missed cleavages?)

‐multiple peptides selected and fragmented

‐in‐source fragmentation resulting in “nonspecific” peptides

‐in‐source water loss (Ser, Thr, Asp, Glu)

‐short peptides may not yield enough fragments for confident ID

‐low quality spectra

‐incorrect monoisotopic m/z

‐incorrect charge state

• proteolytic cleavages

N‐terminal Met cleavage

signal peptide

propeptide

• chemical group

acetylation, phosphorylation, glycosylation, ubiquitination, 

sumoylation, lipidation...

• intra‐ or inter‐ peptidic linkages

disulfide  bonds...

(if) reflected in the molecular weight of the protein and corresponding peptide: amenable to MS usually substoichiometric: requires enrichment of modified  peptides prior to MS modified peptides may feature similar or different   fragmentation pattern: alternative fragmentation  techniques (e.g. ETD for glycopeptides) characteristic fragmentation: pinpointing modified peptides  (carbohydrate oxonium ions for glycopeptides, neutral loss  ions for phosphopeptides / Met‐oxidized peptides)

+80 Da ‐98 Da ‐98 Da_‐98 Da ‐98 Da ‐98 Da ‐98 Da HVG m/z 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 % 0 100 % 0 100

100728_szbk_01 57 (21.531) Cn (Top,4, Ar); Sm (Mn, 2x1.00); Sb (1,40.00 ); Cm (57:58) 2: TOF MSMS 607.97ES+ 7.71e3 184.10 147.13 70.07 139.07 127.10 117.08 357.29 202.11 245.15 312.17 284.17 339.28 501.36 444.34 358.30 443.30 414.28 483.35 445.33 942.64 885.61 701.50 614.46 502.37 511.31 550.32 615.46_616.48 683.50 788.54 702.51 770.52 703.51 789.56 867.59 790.54 822.49 886.62 924.65 906.63 943.63 944.64 1013.69 945.65 1070.69

100728_szbk_02 5 (21.980) Cn (Top,4, Ar); Sm (Mn, 2x1.00); Sb (1,40.00 ); Cm (5:6) 2: TOF MSMS 647.97ES+

174 184.10 70.07 _139.07 127.10 117.08 99.07 483.35 202.11 245.16 357.29 255.14 312.16 284.17 330.18 _426.33 358.30 465.31 867.58 683.52 596.47 581.36 484.35 568.37 608.36 647.47 770.56 694.43 770.48 781.48 850.47 924.63 868.53 868.60 925.63 1022.67 965.59 1052.72

Phosphopeptides fragment similarly to unmodified peptides (CID)

phosphopep

tide

peptide

TPIVGQPSIPGGPVR 

Same peptide glycosylated (HexNAcHexSA) 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bunda nc e 879.6 582.4 880.6 1064.7 578.4 893.6 596.4 1163.8 875.6 312.3 411.3 _{485.4 597.4} 894.6 1007.8 383.4 468.4 695.6 782.6 1277.0 310.2 339.3 561.5 639.0 762.7 858.5 895.6 990.8 1104.8 1146.9 1242.9 080213_02 #2601 RT:33.15 AV:1 NL:1.53E4

T:ITMS + p NSI t d Full ms2 [email protected] [185.00-1435.00]

300 400 500 600 700 800 900 1000 1100 1200 m/z 0 10 20 30 40 50 60 70 80 90 100 R el ati ve A bu ndan ce 921.0 840.0 839.7 738.3 738.0 920.3 921.7 739.3 274.0 292.0 560.0 _656.7 838.7 614.0 453.3 841.3 366.3 492.3 659.0 293.0 411.3 559.0 582.3 630.7 720.0 740.3 833.3 910.0 924.3 965.3 1066.31097.0 1164.3 1212.0 1257.7 b₃ b₄ b₆ b9 y₆ y₉ y₁₁ y₁₂ y₁₃ y₅ y₈* (2+) y8 y10

Sialic acid‐related

oxonium ions

Carbohydrate‐loss related

fragment ions

Glycopeptides show different fragmentation 

Quantitative proteomics 1.

Gel‐based

• 2D gel‐electrophoresis

– time consuming, labor intensive

– limited dynamic range

– not suitable for 

• LMW (15kDa) and HMW (>150 kDa) protein

• hydrophobic (e.g. membrane) proteins

• insoluble proteins

– needs min. 100 g total protein/gel

– 3 replicates / sample

• Improvements

– more sensitive staining

– large format – higher resolving gel

– sample prefractionation

Quantitative proteomics 2.

Gel free –MS based

• Stable isotopic labeling (2_H, 13_C, 15_N, 18_O)

– Chemical labeling

• ICAT (isotope coded affinity tag, Cys)

• iTRAQ, TMT (Multiplexed isobaric tagging technology, Lys/peptide N‐term) 

• ICPL (isotope‐coded protein label)

• Formaldehyde + NaBH₃CN (peptide N‐term, Lys)

– Enzymatic labeling

• 16_O/18_{O exchange catalyzed by trypsin (peptide C‐term)}

– Metabolic labeling

• SILAC (Stable isotope labeling with amino acids in cell culture)

• 15_N or 13_{C labeling (complete or partial)}

• Label‐free LC‐MS quantification

Cell/ 

tissue Protein Peptide

MS

SILAC ICAT iTRAQ, TMT Internal 

Standards

Thank you for your attention!

This work is supported by the European Union, co-financed by

the European Social Fund, within the framework of "

Practice-oriented, student-friendly modernization of the

biomedical education for strengthening the international

competitiveness of the rural Hungarian universities "