Protein Mass Spectrometry (Proteomics) - Ohio … · Protein Mass Spectrometry (Proteomics) ... –- DIGE ˜ Non Gel-Based ... requires clean sample Measure peptides fast experiment,

Protein Mass Spectrometry(Proteomics)

Liwen ZhangMass Spectrometry and Proteomics Facility

The Ohio State University

Summer Workshop

Proteomics:Proteome=Protein + Genomethe large-scale study of proteins, particularly their structures and functions.

Why are proteins important:•The major elements of most cellular structures•Perform most cellular functions•Targets of drugs/toxicants

What Can Proteomics Do

Protein(s) identification

PTM Identification

Protein CrosslinkIdentification

Drug Binding SiteIdentification

Pathway AnalysisProtein Profile Analysis

Protein Interaction Analysis

Traditional Methods for Proteome Research

SDS-PAGEseparates based on molecular weight and/or isoelectric point10 fmol - > 10 pmol sensitivityTracks protein expression patterns

Protein SequencingEdman degradation or internal sequence analysis

Immunological MethodsWestern Blots

SDS-PAGE alone can track the appearance, disappearance or molecular weight shifts of proteins, but can not ID the protein or measure the molecular weight with any accuracy

Edman degradation requires a large amount of protein and does not work on N-terminal blocked proteins

Western blotting is presumptive, requires the availability of suitable antibodies and have limited confidence in the ID related to the specificity of the antibody.

Proteome Research by Mass Spectrometry

Measure M.W. of the protein/peptideSequence amino acid sequence of protein/peptides

High throughputSensitivityAccuracySpecificity

(M+2H)+2

M A S S Sp P E C K

(M+2H)+2

M A S S Sp P E C K

Protein Chemistry

MassSpectrometry

Computing (+ Bioinformatics)

Proteomics by Mass Spectrometry

Protein ID(s)

PTM(s)

Quantitation

Protein Chemistry

•Sample purification

Protein

fractions digest

peptides

•Sample isolation/clean-up

MassSpectrometry

All types of hardware used in proteomics

ESI

MALDI

FT ICR

Ion Trap LTQOrbitrapamaZon

TOF UltraFlexmaXis

Quandrupole Triple-Quad

Computing (+ Bioinformatics)

Molecular weight measurement of the protein/peptideProtein Identification/Confirmation

Protein Identification/PTM Investigation�Global Digestion/LC-MSMS/Bioinformatics�Fractionation/Digestion/LC-MSMS/Bioinformatics

Quantitative Proteomics�Gel-Based Technique

–- DIGE

�Non Gel-Based Techniques–-Isotopic Labeling Techniques:

ITraq/SILAC/18O, 15N. 2H Labeling–-Lable Free Quantitation:

Spectra Counting/Peak Area/EmPAI

MALDI Imaging

What Can Mass Spectrometry Do for Proteomics

Measure proteinPositives:fast experiment, observe changes in protein massNegatives:not good for protein id due to resolution & PTMs, requires clean sample

Measure peptidesPositives:fast experiment, identify proteins & PTMsNegatives:not good for mixtures of proteins, confidence in identification not ideal

Measure fragmentsPositives:identify protein & PTMs, high result confidence.Negatives:longer instrument time, longer database search

TOP DOWN Peptide Fingerprinting Middle DownBottom Up

MS/MS

ENZYME

MS/MS

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

A15A16

A17

A18

A19

A20A21

A14A13

A12

A11

A10

10000 11000 12000 13000 14000 15000 16000 17000 18000 19000 20000mass74

100

%

A

m/z deconvolutesto16952

(M+H)+

(M+2H)+2

15000100005000 m/z

16950

MALDI of Myoglobin

Electrospray of Myoglobin

GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

Sequence of Myoglobin

MW = 16951

Molecular Weight Measurement of the Protein

Protein Identification by Top Down MS/MS

m/z750730710690670650630610590570550

c17+84

c26+70

c26+112

c12+42

c22+112

c12+42

c13+42

c25+112

c23+112

c40+70

c40+112

C24+70

c24+112

c7+42

c15+42

c37+70c55+112

c37+140

c37+112

c38+112

c26+70

c26+112

c52+70

c52+112

c54+70

c54+112

c18+84

z35+14

z35+56

z12

c59+70c59+112

z20

C60+112

m/z750730710690670650630610590570550

c17+84

c26+70

c26+112

c12+42

c22+112

c12+42

c13+42

c25+112

c23+112

c40+70

c40+112

C24+70

c24+112

c7+42

c15+42

c37+70c55+112

c37+140

c37+112

c38+112

c26+70

c26+112

c52+70

c52+112

c54+70

c54+112

c18+84

z35+14

z35+56

z12

c59+70c59+112

z20

C60+112

1000900800700600 780770760750

m/z

+1Ac + 2Me

+1 Ac+ 4Ac

+ 5Ac

+ 2Ac + 2Me

Parents ions (750 - 770, 15+) 758.0864 15+

754.7814 15+

+ 4Ac

- 15+

15+

1000900800700600 780770760750

m/z

+1Ac + 2Me

+1 Ac+ 4Ac

+ 5Ac

+ 2Ac + 2Me

Parents ions (750 - 770, 15+) 758.0864 15+

754.7814 15+

+ 4Ac

- 15+

15+

ECD MS/MS

m/z1000990980970960950940930920910900

z32

z48

z64

z49+14

z9

z65

z49z66 z25+16

z50

z34

z67

z76

z42

z42+14

z59

z51

z68

z43

z78z61 z79

z62

z27z75

m/z1000990980970960950940930920910900

z32

z48

z64

z49+14

z9

z65

z49z66 z25+16

z50

z34

z67

z76

z42

z42+14

z59

z51

z68

z43

z78z61 z79

z62

z27z75

Histone H4

N-AGRGK5GGK8GLGK12GGAK16RHRK20VLRDNIQGIT H4

Ac Ac Ac Ac AcMe Me

Ac Ac Ac

Known Modification

Modification Detected

Protein Identification by Top Down MS/MS

Middle Down

Why Middle Down:simplifies the complex mixtures offered by bottom up strategieswhile avoiding the diminished performance of top down experiments

GluC Cleavage

GluC Cleavage GluC Cleavage

GluC Cleavage

GluC Cleavage GluC Cleavage GluC Cleavage

GluC Cleavage

m/z Sequence 924.5149 43ISLSFTTR50

1050.5439 31APTVHGGAGGAR42

1078.6255 83LASYLEKVR91

1090.5375 374LLEGESEGTR383

1095.5905 284QGDIHELKR292

1108.6374 104ILKWHQQR111

1244.5477 51SCPPPGGSWGSGR63

1360.7141 235EDLIKVLEDMR245

1390.6995 92ALEEANMKLESR103

1494.8097 410LVLCQVNEIQKHA422

1584.9220 246QEYELIIKKKHR257

1972.9869 64SSPLLGGNGKATMQNLNDR82

2277.1279 181TLDNLTIVTTDLEQEVEGMR200

2673.2124 1MNSGHSFSQTPSASFHGAGGGWGRPR26

2790.4785 350QNNEYQVLLGIKTHLEKEITTYR372

2833.5088 384EESKSSMKVSATPKIKAITQETINGR409

2856.3543 322YSCKLQDMQEIISHYEEELTQLR344

2870.3261 258DLDTWYKEQSAAMSQEAASPATVQSR283 3332.6315 293TFQALEIDLQTQYSTKSALENMLSETQSR321 3368.6467 152MAVDDFNLKYENEHSFKKDLEIEVEGLR179

Bottom Up

Data Analysis for Peptide Mass Mapping

• Important data– multiple peaks– mass accuracy– confirming information

(pI, approx. mass, organism, etc.)

?MS

MS Peptide MWFound in Selected

DatabasesNDALYFPT...SWDLTAL...

PTDLDVSY...

protein peptides identify

rank

Cut protein into manageable pieces for the mass spectrometer to analyze.

mass position peptide sequence

927.486 161-167 YLYEIAR

1050.485 588-597 EACFAVEGPK

1282.703 361-371 HPEYAVSVLLR

1305.708 402-412 HLVDEPQNLIK

1439.504 360-371 RHPEYAVSVLLR

1478.788 421-433 LGEYGFQNALIVR

1567.735 347-359 DAFLGSFLYEYSR

1639.931 437-451 KVPQVSTPTLVEVSR

1667.893 469-482 MPCTEDYLSLILNR

1823.892 508-523 RPCFSALTPDETYVPK

2045.021 168-183 RHPYFYAPELLYYANK

2506.243 469-489 MOXPCTEDYLSLILNRLCVLHEK

1 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL71 TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK141 PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE211 KVLTSSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR281 ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL351 GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK421 LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT491 PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP561 KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA

Bovine Albumin

mass position peptide sequence

927.486 161-167 YLYEIAR

1050.485 588-597 EACFAVEGPK

1283.703 361-371 HPEYAVSVLLR

1305.708 402-412 HLVDEPQNLIK

1439.504 360-371 RHPEYAVSVLLR

1479.788 421-433 LGEYGFQNALIVR

1567.735 347-359 DAFLGSFLYEYSR

1639.931 437-451 KVPQVSTPTLVEVSR

1667.893 469-482 MPCTEDYLSLILNR

1823.892 508-523 RPCFSALTPDETYVPK

2045.021 168-183 RHPYFYAPELLYYANK

2506.243 469-489 MOXPCTEDYLSLILNRLCVLHEK

YLY

EIA

R

EAC

FAVE

GPK

HP

EYA

VSV

LLR

LGE

YG

FQN

ALI

VR

RH

PYFY

APEL

LYYA

NK

RPC

FSAL

TPD

ETYV

PK

Peptide Mass Mapping

MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVAD ESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP ERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLK KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAA CLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV ARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVF LGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVP QVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKC CTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV AASQAALGL

Human Albumin

m/z Missed Cleavages Sequence

1013.4244 0 589ETCFAEEGK597

1013.5990 0 599LVAASQAALGL609

1141.5194 1 589ETCFAEEGKK598

1141.6939 1 598KLVAASQAALGL609

1149.5759 1 25DAHKSEVAHR34

1149.6150 0 66LVNEVTEFAK75

1352.6661 1 497VTKCCTESLVNR508

1352.7685 1 427FQNALLVRYTK437

1623.7876 0 348DVFLGMFLYEYAR360

1623.9581 1 362HPDYSVVLLLRLAK375

1898.9952 1 169RHPYFYAPELLFFAK183

1898.9952 1 170HPYFYAPELLFFAKR184

0

MS alone CAN NOT distinguish peptides of similar molecular weight.

post-translational modification.sequence coverage determinations difficult.the identification of more than one protein in a sample impossible.Mass accuracy requirements

Mixture of Peptides

MS Analyzer

Retention Timea b c

m/z a

m/z b

m/z cCollision induced dissociation

(CID)

LC/MS or LC/MSMSPeptides with different m/z

Tandem (MS/MS) – Peptide Fragment Analysis

# b Seq. y #1 58.0287 G 132 171.1128 L 1362.7515 123 284.1969 L 1249.6674 114 413.2395 E 1136.5834 105 528.2664 D 1007.5408 96 641.3505 L 892.5138 87 698.3719 G 779.4298 78 861.4353 Y 722.4083 69 976.4622 D 559.3450 5

10 1075.5306 V 444.3180 411 1174.5990 V 345.2496 312 1273.6674 V 246.1812 213 K 147.1128

GLLEDLGYDVVVK

CASP4_MOUSECaspase-4OS=Mus musculus

LC/MSMS

MSMS Fragments and Different Fragmentation Technique

CID: a, b and y ion, loss of H2O, NH3, side-chain PSD: a, b and y ionIRMPD: b and y ion, loss of side-chainECD: c, y and z ionsETD: c, y and z ions

H2N C CO

NH

R1

HC C

ONH

R2

HC C

ONH

Rn-1

HC C

ORn

OH…

b1 a2 b2 c2 an-1 bn-1 cn-1

x1 y1 z1xn-2 yn-2 zn-2xn-1 yn-1

c1

zn-1

a1

b3b2b1

y1y2y3

1598

14241295

1166

1052

965852

723

586529

401Q G H E L S N E E R

Sequence Nomenclature for Mass Ladder

b2‐b1=56Da + R2 b3‐b2=56Da + R2

y3‐y2=56Da + R2 y2‐y1=56Da + R2

XX

Mass=56Da+R

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

246.00y2

345.32y3

444.29y4

559.27y5

722.22y6

892.36y8

1007.39y9

1136.48y10

1249.63y11

779.16y7

283.82b3

413.06b4

528.07b5

641.08b6

698.27b7

976.45b9

1075.51b11

1174.68b12

1273.72b13

861.35b8

99.32Val

98.97Val

114.98Asp

162.95Tyr

56.94Gly

113.20Leu

115.03Asp

129.09Glu

113.19Leu

99.17Val

99.06Val

115.10Asp

163.08Tyr

57.19Gly

113.01Leu

115.01Asp

129.24Glu

99.04Val

G L L E D L G Y D V V V Kb12b10 b11b7 b9b4 b5b3 b6 b8

y4 y2y5y6y8y10 y9y11 y7 y3

Data Dependent Top5 Method

Data Dependent Acquisition and Dynamic Exclusion 30143_WT_LEG #14457-14668 RT: 88.19-89.24 AV: 19 NL: 3.22E5T: FTMS + c ESI Full ms [350.00-2000.00]

400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

743.89

649.66

997.60

736.42

499.30 729.64

584.62

758.15

562.36 643.41 788.92

445.12958.96

601.571063.19

947.46417.92 1044.181163.58538.31 668.66 876.43683.33

991.52

837.38480.61639.64

912.40817.43

1007.50

1193.31

508.31

1077.51 1154.23

724.35

1

32

54

67

8

MSMS

30143_WT_LEG #14664 RT: 89.25 AV: 1 NL: 1.39E3T: ITMS + c ESI d Full ms2 [email protected] [190.00-2000.00]

400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

898.13712.51

991.16

962.47776.52

918.39926.34

878.32589.17

638.90560.70

869.18734.69

525.26 693.22468.12 766.40 816.38

850.10660.95 1019.05

1048.37617.69

1167.301120.23424.27454.82

1092.28


400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

907.18

945.07624.99

738.98 850.52950.34

497.15722.80 767.95 1037.29898.17693.97 1167.831066.54

963.19833.56468.92 1136.33668.46601.35 1196.351022.35 1101.94418.44 511.27 579.50


400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

622.91

679.63

515.34

819.15

557.39

510.16

715.31651.39566.39 1001.25

466.65574.64

932.45

540.92409.65 724.31501.05420.13 611.22 809.47 1019.31 1132.30737.84 1100.22793.33 873.75


400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1002.30

648.24

761.20 824.14

644.32

874.36711.34

1158.45

1097.28727.35 984.16470.06

442.20 598.03487.98

693.59805.93580.22509.03 622.06

562.69 919.63795.91 1193.021080.75868.80 967.13 1118.91424.11 1068.94


400 450 500 550 600 650 700 750 800 850 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

546.11

762.93 817.42

417.30 617.37 696.97878.39

706.20746.46

660.79

711.89822.64

842.66

489.08 869.10

641.63 807.75 854.16674.05 725.63

799.31881.19566.84 599.46540.10

580.62 886.21526.61

432.28

12 3

45

MS Cycle: 0.06SLC Peak Width >12S

Dynamic Exclusion

Make MSMS for the lower abundant ions possible!!

VFGTDMDNSR

IFDDSDQTK

LVNLGK

QAEDVNLLDQMSK

If all of these peptides belonged to anunknown protein, MS/MS couldpotentially reveal protein identity

More Peptides IdentifiedIncrease Confidence in ID

Data Analysis for MS/MS Method

?MS/MS

MS Peptide MWFound in Selected

DatabasesNDALYFPT...SWDLTAL...PTDLDVSY...

protein peptides identify

rank

200 400 600 800 1000 1200 1400 1600

Rel

ativ

e In

tens

ity

m/z

theoretical spectra200 400 600 800 1000 1200 1400 1600

0

20000

40000

60000

80000

100000

120000

Rel

ativ

e In

tens

ity

m/z

compare

Database SearchingMascot•16 node cluster for high-speed data processing

Protein sequences are digested and fragmented In Silico which produces an enormous peak list

Raw MS/MS data is converted to a peak list and compared against the In Silico peak list.

Critical point of database searching•The enzyme must work properly (no non-specific cleavage and missed cuts)•The mass accuracy limits must be set appropriately•Any modifications must be accounted for (modified cysteine)•The database must contain the protein

Mascot Database Search Engine

20ppm for orbitrap1.5 Da for LTQ

Enzyme Used

MASCOT Results

Protein Name

Protein Mascot Mowse Score

Protein Mol. Weight

# of spectra matched# of peptides matched

The Exponentially Modified Protein Abundance Index (emPAI)

MASCOT Results

*

*

Peptide Sequence and MS/MS Information

Protein Name

MASCOT Results

*

Peptide Sequence and Sequence Coverage

MASCOT Results

Protein Report

Post-translational Modifications

Modification Modified residue Mass Shift (Da)

Disulphide bond formation C -2.0157Deamidation of Asn and Gln Q/N 0.9840Methylation K/R 14.0157Hydroxylation P 15.9949Oxidation of Met M 15.9949Acetylation K 42.0106Carboxyamidomethylcysteine(iodoacetamide) C 57.0215

Phosphorylation S/T/Y 79.9663Biotinylation (amide bond to lysine) K 226.0776Nitration Y 44.9851Ubiquitination K 114.0429Oxidation of Cys C 31.9721/47.9847nitrosylation C 28.9902

Peptide Modifications

• Virtually anything that shifts the mass can be determined by MS

• Using MS/MS allows for identification of the modification AND location

• You must have an idea of what modification you are looking for

• First use protein stain to examine various modifications (multiplex analysis)

• Western analysis VS MS analysis

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z

712.632+

y14

753.582+

b14

276.20y2

347.18y3

446.27y4

547.27y5

618.41y6

717.41y7 774.38

y8

911.36y9 1109.47

y11

1166.53y12

1295.49y13

1423.55y14

1010.47y10

357.19b2

486.30b3

543.30b4

642.31b5

741.36b6

878.49b7

935.48b8

1034.45b9

1206.44b11

1305.59b12

1376.51b13

1505.59b14

1105.51b10

44X K E G V V H G V A T V A E K60

X=C10H16N2O4 (228.1105 Da)

Theoretical m/z 826.45432+

Observed m/z 826.45732+

Mass Error 3.63 ppm

y4 y2

b13 b14

y5

b12

y6

b10 b11

y8y10 y9

b7 b9

y11y12

b4 b5

y13

b3 b6 b8

y7

b2

y3y14

70.98A

99.09V

101.00T

71.13A

99.00V

56.97G

136.98H

99.11V

99.00V

57.06G

128.96E

128.06K

70.92A

99.15V

100.93T

71.06A

97.97V

56.99G

137.13H

99.05V

99.01V

57.00G

129.11E

129.98E

Mass of X=(M+H)-y14=826.4543*2-1-1423.55=228.35

500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

638.892+

b8

638.312+

y9 1274.57y9

738.122+

b10

794.642+

b11

795.572+

y11

960.47y7

573.11b4

759.32b5

906.56b6

1062.49b7

1276.67b8

1474.67b10

186.21Trp

97.24Phe

155.93Arg

214.18(103+111)C-DMPO

198.00 (97+101)Pro-Thr

900E E W K W F R C907(DMPO) P T L L911

b4 b5 b6 b7 b8 b10 b11

y11 y9 y7

Theoretical m/z=859.942+

Observed m/z=859.892+

DMPO=C6H9NO (111.0684 Da)

DMPO Adduct

300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

1500

317.28y3

404.24y4

503.32y5

617.36y6

718.37y7

817.37y8

984.39y9

1083.45y10

1197.51y11

1311.59y12

1426.56y13

391.12b4

505.27b5

619.29b6

718.37b7

1484.46b14

984.39b9

1085.46b10

1199.51b11

1298.46b12

1385.54b13

886.36y9*

1099.56y11*

1213.70y12*

886.36b9*

1101.51b11*

1200.51b12*

1287.76b13*

1386.63b14*

1457.78b15*

Ser Val Asn Thr Val

167.02 (87+80)Ser(PO4)

Val Asn Asn Asp

SerValAsnThr Val

266.02 (87+80+99)Ser(PO4)-ValValAsnAsn

41T S S D N N V S(PO4)48 V T N V S V A K56

bn*=bn-H3PO4yn*=yn-H3PO4m/z=850.542+

802.432+

M-H3PO4

b13b13*b4 b6 b7 b15*

b11b11*b5

y9y9*

y10 y3y4y5y6y8 y7

b14b14*

y11y11*

y12y12*

y13

b10b12b12*

b9b9*

Phosphorylation

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

241E A F P A Y246(N2O) R Q P P E S L253

b3 b4 b7 b8 b11

y3y4y8y9y10y11

219.15y2

348.10y3

445.22y4

542.31y5

670.43y6

826.20y7

1034.50y8

1105.51y9

1202.57y10

1349.69y11

675.552+y11

348.10b3

445.22b4

1105.51b9

1202.57b10

880.42b7

1008.45b8

1331.59b11

601.992+b10

666.502+b11 709.872+

b12

Glu Pro Pro Gln208.3 (163+45)

Tyr(N2O)Arg Ala Pro Phe

GluPro ProGlnPro

b9 b10 b12

y2y6y7 y5

Measured m/z = 775.612+Theoreticalm/z = 775.372+

Nitration

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

460.24y4

623.30y5

736.40y6 823.41

y7

910.39y8

1023.55y9

1151.56y10

1280.54y11

1431.51y121488.48

y13

1617.48y14

1745.66y15

1858.62y16

930.162+

y16

1014.182+

b19

979.482+

y17

584.26b6

713.38b7 770.40

b8921.28

b9

1050.21b10

1178.34b11

1291.39b12 1378.34

b13

1465.42b14

1578.48b15

1741.58b161798.75

b17

1913.59b18

873.542+

y15

E G

150.88(103+48)C(SO3) E K I S S L Y G D

EGEKISSLY Q L

529V G S V L Q E G C(SO3) E K I S S L Y G D L R548y4y5y7y8y9y10y11y12y14

b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 b16

y6y13y15y17 y16

b17 b18 b19

150.97(103+48)C(SO3)

Cysteine Oxidation

Generating Peptides containing modified residue --Different Enzymes

Low population of modifications--Enrichment--Targeted Method

Modification not stable--Experiment Condition (reducing reagents, enzymes)--Fresh sample

Modification labile in MS analysis--Neutral Loss Method--Different Fragmentation Method

Difficulties for PTMs Identification

Trypsin K-X and R-X

Endoprotease Lys-C K-X except when X = P

Endoprotease Arg-C R-X except when X = P

Endoprotease Asp-N X-D

Endoprotease Glu-C E-X except when X = P

Chymotrypsin L-X, F-X, Y-X and W-X

Cyanogen Bromide X-M

Enzymes for Proteome Research

1 MAALKLLSSG LRLGASARSS RGALHKGCVC YFSVSTRHHT KFYTDPVEAV 51 KDIPNGATLL VGGFGLCGIP ENLIGALLKT GVKDLTAVSN NAGVDNFGLG

101 LLLRSKQIKR MISSYVGENA EFERQFLSGE LEVELTPQGT LAERIRAGGA 151 GVPAFYTSTG YGTLVQEGGS PIKYNKDGSV AIASKPREVR EFNGQHFILE 201 EAITGDFALV KAWKADRAGN VIFRKSARNF NLPMCKAAGT TVVEVEEIVD 251 IGSFAPEDIH IPKIYVHRLI KGEKYEKRIE RLSLRKEGDG KGKSGKPGGD 301 VRERIIKRAA LEFEDGMYAN LGIGIPLLAS NFISPNMTVH LQSENGVLGL 351 GPYPLKDEAD ADLINAGKET VTVLPGASFF SSDESFAMIR GGHVNLTMLG 401 AMQVSKYGDL ANWMIPGKMV KGMGGAMDLV SSSKTKVVVT MEHSAKGNAH 451 KIMEKCTLPL TGKQCVNRII TEKGVFDVDK KNGLTLIELW EGLTVDDIKK 501 STGCDFAVSP NLMPMQQIST

Tryspin: 309AALEFEDGMYANLGIGIPLLASNFISPNMTVHLQSENGVLGLGPYPLK356Chymotrypsin: 329ASNF332, GluC: 315DGMYANLGIGIPLLASNFISPNMTVHLQSE344

Phosphorylation

Using the CORRECT Enzyme




PTM Enrichment Techniques (Start with mg of samples)

Acetylation: Anti-acetyl lysine polyclonal antibody

Phosphopeptides: Immobilized Metal Affinity Chromatography (Fe3+)Metal Oxide Affinity Chromatography (TiO2, ZrO2)Reversible Covalent Binding

(Dunn JD1, Reid GE, Bruening ML, Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry. Mass Spectrom Rev. 2010 Jan-Feb;29(1):29-54.)

Phospho-Threonine Antibody (P-Thr-Polyclonal)

Nitration: Anti‐Nitrotyrosine polyclonal antibody

Ubiquitination: K-ε-GG–specific antibody (enrichment has to be done prior to other treatment on lysine)

Glycopeptides: Lectin affinity enrichmentCovalent InteractionsChromatographic separation

Ongay S1, Boichenko A, Govorukhina N, Bischoff R., Glycopeptide enrichment and separation for protein glycosylation analysis., J Sep Sci. 2012 Sep;35(18):2341-72.

Methylation: Anti-methyl lysine/araginine antibody

RT: 10.04 - 89.85

15 20 25 30 35 40 45 50 55 60 65 70 75 80 85Time (min)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

35.88

42.9541.90

49.23

49.07

40.05

45.5136.2769.9828.36

61.2660.55

65.79 71.3953.2834.20

59.1532.0427.80

61.7524.09 74.1717.6789.7880.23 82.6574.6210.19 14.63

44.39

35.70

65.64 69.7936.07

42.6641.65 44.57

39.72

53.0348.95 61.7528.19 61.2549.11 70.1849.40

57.3134.12 71.3327.61 65.82

17.77 74.1723.8680.19 82.5578.61 89.7314.23

NL:2.47E8Base Peak MS 17625_1_Rerun

NL:3.98E8Base Peak MS 17625_1_target_1

Full Scan

Scan for 479.27302+ (SLVLfGlyTPSR ) and 516.77932+ (SLVLC(CAM)TPSR)

17625_1_target_1 #1211-2280 RT: 27.51-40.35 AV: 25 NL: 4.96E2T: ITMS + c ESI d Full ms2 [email protected] [120.00-1450.00]

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

581.20

359.28

470.05

460.31757.37417.14

658.29545.37

413.03601.33

300.11201.06 670.45403.45286.23173.18 448.88

640.46255.07 487.34 730.46337.35219.26 511.32 812.38 833.42687.02 782.91

No MSMS spectrum for 479.29302+

Targeted MS Scan (for Known Modifications)

SLVLfGlyTPSR

99Val

113Leu

85fGly

101Thr





m/z400 600 800 1000 1200 1400 1600250

Chain A

Chain B

185N A C187 G S G Y D F D V F V V R199

138L V E G C142 L V G G R146

S

S

408.17 465.20 552.31 609.30 772.30 1034.35 1149.39 1248.41 1395.53 1494.50 1593.59

289.23y3B

342.33b3B

501.10y5B

274.25y2A

373.31y3A

520.42y4A

619.48y5A

734.51y6A

1303.64y11A

1159.62y9A

1216.57y10A

1360.66y12A

996.57y8A

y2Ay3Ay4Ay5Ay6Ay8Ay10Ay11Ay12A y9A

V F V D FD Y G S G

VFVDFDYGSG V

y3By5B

b3B

A

B

Adjust Experiment Condition to Preserve Modifications

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

194A G R P G M G V O202 G P E T S L208

y4y5y7y8y9y10y12

b3 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14

y6 y3

Theoretical m/z =783.40892+, Observed m/z =783.40802+, Mass Error =1.15ppm

674.472+b13

285.21b3

439.23b5

570.30b6

627.43b7

1020.49b10

726.35b8

963.39b9

1117.55b11

1347.71b13

1246.54b12

718.062+b14

623.822+b12

154.13 (97+57)P‐G

130.97M

57.13G

98.92V

237.04O

101.17T

57.10G

97.06P

128.99E

320.13y3

449.05y4

546.24y5

603.21y6

840.38y7

939.24y8

996.30y9

1127.37y10

1281.45y12

128.92E

97.19P

56.97G

237.17 O

98.86V

57.06G

131.07M

154.08 (57+97)G‐P

O=Pyrrolysine

Using Different Enzyme to Protect Modifications




Modification labile in MS analysis--Neutral Loss Method--Different Fragmentation Method


Scan ScanDissociate

Neutral Loss Scan

Phosphopeptides can lose phosphoric acid on CID (Serine, Threonine)

+ Pi(H3PO4)

98 amu Neutral (M+H)+

49 amu Neutral (M+2H)2+

LDIFpSDFGGLK

98//32.7

113

129

Neutral Loss Scan

Image from http://www.mcponline.org/content/9/3/579/F5.expansion

ECD/ETD: Keep the Phosphorylation Intact

PO3 or H3PO4 groups may leave upon CID activation

actual amino acid location of PO3 group may not be clear

Electron Capture Dissociation (ECD) requires an ICR; Electron Transfer Dissociation (ETD) in traps

• Peptide fragments without loss of phospho group

Sweet, Anal. Chem. 2006, 78, 7563-7569

Leann M. Mikesh et al. Biochimica et Biophysica Acta 1764 (2006) 1811–1822

Identification of PTMs:

High Mass Accuracy

High Samples Purity

High Concentration

Know the Modification (Stability, Mass Shift, possible modification site)

Protein Complex Identification(Whole cell lysate, IP Products)

Clean Sample!!!(Less Salt, No Detergent

Lipids, DNA Free, Keratin Free)

Better SeparationMore sensitive Instruments

On Bead DigestionPre-Fractionation

Method Separation based on

Separation done using Further steps

Gel Electrophoresis (1D) Molecular mass

Gel (which acts like a molecular sieve) and potential

In-gel digestion of proteins to peptidesLC-MS/MSGel Electrophoresis

(2D)

Isoelectric point (pI; IEF) & Molecular mass

Gel, potential and ampholytes

Reverse Phase (C8 or C4) chromatography

Combination of hydrophobicity and molecular weight

HPLC

Protein(s) Digest to peptides LC-MS/MS

Gel Filtration Molecular Weight HPLC

Ion Exchange Cation or Anion affinity FPLC

Affinity Chromatography DNA,RNA, Anti-body, peptides etc

HPLC

Types of Separation Technologies for Molecules

Pre-Fractionation Method

LC/MSMSLC/LC/MSMS

High Abundant Protein Depletion1D SDS FractionationOff-gel Fractionation

SCX Fractionation

1

2

3

4567

8

9

10

11

In Gel Digestion

In Gel Digestion

In Gel Digestion

In Gel DigestionIn Gel DigestionIn Gel DigestionIn Gel Digestion

In Gel Digestion

In Gel Digestion

In Gel Digestion

In Gel Digestion

Same Volume

LC/MSMS

LC/MSMS

LC/MSMS

LC/MSMSLC/MSMSLC/MSMSLC/MSMS

LC/MSMS

LC/MSMS

LC/MSMS

LC/MSMS

MASCOT Search

1D SDS PAGE Fractionation LC/MSMS Workflow

>1800 Proteins identifiedGlobal Digestion: 920 ProteinsSCX‐LC/MSMS: 981 Proteins1D SDS‐LC/MSMS: >1800 Proteins

Quantitative ProteomicsQuantitation in proteomics has become a popular area in recent proteomics research with the development of quantitation techniques such as DIGE, SILAC, ICAT, iTRAQ and Label Free.

•Difference Gel Electrophoresis•Gel based using cy-dye chemistry.

•Isobaric tag for relative and absolute quantitation – iTRAQ• is a non-gel based technique used to identify and quantify proteins/peptides from different sources in one single experiment by using isotope coded covalent tags that will label the N-terminus and side chain amines of peptides from protein digestions.

•Stable isotope labeling by amino acids in cell culture – SILAC• is a non-gel based approach for in vivo incorporation of a label into proteins for MS quantitative proteomics. It relies on metabolic incorporation of a given 'light' or 'heavy' form of the amino acid into the proteins.

•Label Free Quantitation•It has been observed the chromatographic peak areas and number of spectra/peptides observed for a protein in a LC/MS/MS run is correlated with the concentration of that particular protein.

DIGE (Difference Gel Electrophoresis)

Spot Volume = [spot 1 on treated]/[spot 1 on standard]


In Gel Digestion, LC/MSMS, MASCOT


high sensitivity

linearity of the dyes utilized

straightforward significant reduction of experiment error

High reproducibility

Pros and Cons to DIGE

300 – 500 µg of total protein is required for each biological replicate

Requires high resolution 2D gels

Not ideal for membrane proteins

Not ideal for serum type samples

Some protein spots identify more than one protein or do not have enough protein to identify the spot

Labor Intensive

Overview of iTRAQ™ Reagents Methodology

Labeling Lys and N‐terminus

Up to 8 samples

Fractionation offers more identification

Trypsin digestion

Cell Culture + Tissue

Isobaric tag for relative and absolute quantitation

Chemistry of TMT™ Reagents

Figure Provided Courtesy of Thermo Fisher Scientifics

Figure Provided Courtesy of Thermo Fisher Scientifics

Workflow of TMT™ Reagents

Group IS Normal Mod Mild Mix1 IS 1 (126) 2004 (127) 2041 (128) 2043 (129) 2069 (130)2 IS 2 (126) 2007 (131) 2042 (127) 2040 (128) 2066 (129)3 IS 3 (126) 2008 (130) 2065 (131) 2070 (127) 2037 (128)4 IS 4 (126) 2063 (129) 2046 (130) 2038 (131) 2014 (127)5 IS 5 (126) 2015 (128) 2068 (129) 2071 (130) 2036 (131)6 IS 6 (126) 2016 (127) 2067 (128) 2062 (130) 2035 (131)

Dry Eye Studies: 4 Groups: Normal, Moderate Dry Eye, Mild Dry Eye, Mixed Dry Eyes

100 300 500 700 900 1100 1300 1500m/z 19001700

232.12y2

331.31y3

559.25y5

674.35y6

821.55y7

968.30y8

1131.44y9

1245.38y10

1344.23y11

1401.32y121458.29

y131605.40y14

1706.27y15

1834.52y16

487.24b2

588.18b3

792.12b5 849.14

b6948.30b7

1062.54b8

1225.15b9

1372.42b10

1519.43b11

1634.48b12

1733.70b13

1862.61b14

735.20b4

359.16b1

Peptide Sequence:EQTFGGVNYFFDVEVGRProtein Name : Human Cystatin-S

Observed m/z= 1097.02702+

Theoretical m/z= 1097.04432+

124 126 128 130 132 134m/z05

101520253035404550556065707580859095

100

Rel

ativ

e A

yund

ance 126

127 129128

131Ratio 126:127:128:129:130:131=1:0.30:0.09:0.25:0.00:1.49

99.17Val

227.94 (129+99)Glu-Val

115.10Asp

147.20Phe

146.75Phe

163.14Tyr

113.94Asn

98.85Val

57.09Gly

56.97Gly

147.11Phe

100.87Thr

128.25Gln

99.22Val

115.05Asp

147.01Phe

147.27Phe

162.61Tyr

114.24Asn

99.16Val

56.92Gly

57.02Gly

147.02Phe

100.94Thr

128.08Gln

128.91Glu

iTraq MASCOT Results

Protein Name

DE/NDE (Ratio (# of Detection, p value))

FunctionMild/Normal Mod/Normal Mix/Normal

Aldehyde dehydrogenase, dimeric NADP-preferring OS=Homo sapiens (2.41, 6, 0.0030) (1.69, 6, 0.0471) oxidoreductase activity

Apolipoprotein A-I OS=Homo sapiens (1.75, 6, 0.0362) transporter activity; protein binding; enzyme regulator activity; lipid binding

Cystatin-S OS=Homo sapiens (0.51, 6, 0.0014) (0.43, 6, 0.0005) (0.60, 6, 0.0283) enzyme regulator activity

Deleted in malignant brain tumors 1 protein OS=Homo sapiens (0.60, 6, 0.0472) signal transducer activity; protein binding; bacterial cell surface binding

Ezrin OS=Homo sapiens (1.79, 6, 0.0101) protein binding; binding

Hemopexin OS=Homo sapiens (2.15, 6, 0.0384) (2.11, 6, 0.0486) binding; transporter activity; ion binding

Haptoglobin OS=Homo sapiens (1.97, 6, 0.0474) catalytic activity; protein binding; peptidase activity

Ig alpha-1 chain C region OS=Homo sapiens (0.59, 6, 0.0113) antigen binding; protein binding

Ig gamma-1 chain C region OS=Homo sapiens (1.94, 6, 0.0266) antigen binding; protein binding

Ig gamma-2 chain C region OS=Homo sapiens (1.87, 6, 0.0369) antigen binding

Ig gamma-3 chain C region OS=Homo sapiens (2.08, 6, 0.0408) antigen binding

Extracellular glycoprotein lacritin OS=Homo sapiens (0.28, 6, 0.0059) (0.23, 6, 0.0003) (0.48, 6, 0.0428) protein binding; extracellular matrix binding

Putative lipocalin 1-like protein 1 OS=Homo sapiens (0.43, 6, 0.0026) (0.39, 6, 0.0002) (0.50, 6, 0.0119) binding; transporter activity

Lipocalin-1 OS=Homo sapiens (0.33, 6, 0.0030) (0.32, 6, 0.0003) (0.43, 6, 0.0188) binding; transporter activity; protein binding; enzyme regulator activity

Lysozyme C OS=Homo sapiens (0.33, 6, 0.0019) (0.28, 6, 0.0031) catalytic activity; hydrolase activity; protein binding

Polymeric immunoglobulin receptor OS=Homo sapiens (0.51, 6, 0.0290) (0.43, 6, 0.0016) protein binding

Prolactin-inducible protein OS=Homo sapiens (0.36, 6, 0.0004) (0.38, 6, 0.0008) (0.58, 6, 0.0409) protein binding

Proline-rich protein 1 OS=Homo sapiens (0.44, 6, 0.0023) (0.39, 6, 0.0056) enzyme regulator activity

Proline-rich protein 4 OS=Homo sapiens (0.41, 6, 0.0042) (0.34, 6, 0.0081)

Secretoglobin family 1D member 1 OS=Homo sapiens (0.28, 6, 0.0032) (0.21, 6, 0.0006) (0.41, 6, 0.0169) binding

Mammaglobin-B OS=Homo sapiens (0.34, 6, 0.0076) (0.26, 6, 0.0009) (0.43, 6, 0.0215) steroid binding; binding; hormone binding

Serotransferrin OS=Homo sapiens (2.02, 6, 0.0429) protein binding; ion binding

Lactotransferrin OS=Homo sapiens (0.33, 6, 0.0019) (0.34, 6, 0.0072) peptidase activity; ion binding; pattern bindingcarbohydrate binding; hydrolase activity; protein binding

Vitamin D-binding protein OS=Homo sapiens (1.65, 6, 0.0449) (1.87, 6, 0.0206) transporter activity; steroid binding; vitamin binding;: protein binding

Zinc-alpha-2-glycoprotein OS=Homo sapiens (0.40, 6, 0.0047) (0.36, 6, 0.0029) (0.59, 6, 0.0502) lipid binding; carboxylic acid binding; hydrolase activity; transporter activity

Zymogen granule protein 16 homolog B OS=Homo sapiens (0.44, 6, 0.0383) carbohydrate binding

iTraq Results

Pros and Cons to iTRAQ

Requires less total protein than DIGE (50 µg or less)

One experiment can determine fold change, protein ID and Post translational modification

Up to 8-10 groups can be compared at the same time

Samples can come from both tissue or cell culture

• Mass Spectrometry requirements are rigorousHigh resolutionAdvanced LC chromatographyLong instrument timePQD/HCD needed on IT instrument

• Produces enormous amount of data

• Requiring detailed bioinformatic analysis

• Expensive

Regular Media (Lys Light)

Lys-depleted Media(Lys Heavy)

No Drug Drug Treatment

Combination/Extraction

Digestion/Peptide Identification

Change =

Digestion

Shotgun Proteomics

SILAC (Stable isotope labeling by amino acids in cell culture)

Heavy Isotopic Labeled Amino Acids:

L-Lysine: 13C6, (+6Da), 13C6/15N2 (+8Da), 13C6/15N2/D9 (+17Da), 15N2/D9 (+11Da), D4 (+4Da)

L- Arginine: 13C6 (+6Da), 13C6/15N4 (+10Da), 13C6/15N4, D7 (+17Da),15N4/D7 (+11Da)

L-Tyrosine: 13C9 (+9Da)

Fractionation/IP LC/MSMS

Optimization of Heavy Amino Acids IncorporationS G R G K5 G G K8 G L G K12 G G A K16 R H R K20 V L R D N I Q G I T K31 P A I R R

L A R R G G VK44 R I S G L I Y E E T R G V L K59 V F L E N V I R D A V T Y

T E H A K77 R K79 T V T A M D V V Y A L K91 R Q G R T L Y G F G G

∆ M =4*11= 44

11353

11396

11309

11279

11351

11393

11200 11300 11400 11500 11600

Mass

17881_13Days #21693-22317 RT: 127.45-130.89 AV: 100 NL: 6.03E5T: FTMS + c ESI Full ms [350.00-2000.00]

920 940 960 980 1000 1020 1040 1060 1080 1100m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

935.19

944.68

1012.47

907.49

1082.79984.88

1044.02

945.93

982.84908.801086.07

1014.48

1047.03948.171001.85966.48 1057.00 1064.50924.47914.92

1032.53

1078.08

1041.70

957.65 979.78 1091.02

930.44 1009.11

1023.84

994.55

987.00

1095.56

K* =13C6/15N2 labeled LysR* =13C6/15N4 labeled Arg

Cell Culture Labeled for 7 days

Cell Culture Labeled for 7 days

17872_1_7 #1858-1959 RT: 22.95-23.41 AV: 13 NL: 5.19E4T: FTMS + p ESI Full ms [350.00-2000.00]

660 662 664 666 668 670 672 674 676 678m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

663.38

672.39

663.88

672.90

664.39

673.40

675.40671.89

675.90664.89673.90667.18 670.41661.29

674.88

662.83 676.40 679.39668.18665.35 678.37666.42 668.89

17872_1_7 #1902 RT: 23.14 AV: 1 NL: 1.01E3T: ITMS + c ESI d Full ms2 [email protected] [170.00-1340.00]

200 300 400 500 600 700 800 900 1000 1100 1200m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abund

ance

685.43

606.03456.31

855.50

654.58

492.54 549.04

983.57

742.11511.08 798.47229.90 342.92 584.37

1151.56696.36298.04 428.40358.01 1168.47838.40784.19 1117.35921.24 1027.74279.94 423.46

873.56

615.20

703.42

663.81466.34

1001.27230.07558.07

602.24501.42 816.61342.89725.30

1143.22255.27

983.84825.94775.75 1012.89298.12 411.46

1068.81 1195.44975.96

18Da18Da

∆ M =10+8= 18Da

DNIQGITKPAIR DNIQGITK*PAIR*

K* =13C6/15N2 labeled LysR* =13C6/15N4 labeled Arg

MSMS of DNIQGITK*PAIR* MSMS of DNIQGITKPAIR

Ratio=Heavy/Light=1:1

Accession Description Ratio: Heavy/Light

Sample 1 Sample 2

P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens 0.096 0.049

P22314 Ubiquitin‐like modifier‐activating enzyme 1 OS=Homo sapiens 0.106 0.182

P23526 Adenosylhomocysteinase OS=Homo sapiens 0.250 0.151

P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 0.257 0.265

P52597 Heterogeneous nuclear ribonucleoprotein F OS=Homo sapiens 0.260 0.254

O43175 D‐3‐phosphoglycerate dehydrogenase OS=Homo sapiens 0.262 0.276

P31943 Heterogeneous nuclear ribonucleoprotein H OS=Homo sapiens 0.284 0.337

P23771 Trans‐acting T‐cell‐specific transcription factor GATA‐3 OS=Homo sapiens 0.295 0.379

O00299 Chloride intracellular channel protein 1 OS=Homo sapiens 0.306 0.316

P07741 Adenine phosphoribosyltransferase OS=Homo sapiens 0.319 0.343

Q14103 Heterogeneous nuclear ribonucleoprotein D0 OS=Homo sapiens 0.359 0.390

Q9BQE3 Tubulin alpha‐1C chain OS=Homo sapiens 0.380 0.404

P13639 Elongation factor 2 OS=Homo sapiens 0.391 0.609

P31949 Protein S100‐A11 OS=Homo sapiens 0.394 0.626

P08107 Heat shock 70 kDa protein 1A/1B OS=Homo sapiens 0.412 0.435

P11586 C‐1‐tetrahydrofolate synthase, cytoplasmic OS=Homo sapiens 0.415 0.835

P23246 Splicing factor, proline‐ and glutamine‐rich OS=Homo sapiens 0.418 0.387

P31942 Heterogeneous nuclear ribonucleoprotein H3 OS=Homo sapiens 0.424 0.382

P58546 Myotrophin OS=Homo sapiens 0.429 0.420

P11021 78 kDa glucose‐regulated protein OS=Homo sapiens 0.442 0.514

P00558 Phosphoglycerate kinase 1 OS=Homo sapiens 0.450 0.574

P61978 Heterogeneous nuclear ribonucleoprotein K OS=Homo sapiens 0.472 0.445

P62826 GTP‐binding nuclear protein Ran OS=Homo sapiens 0.475 0.496

P35080 Profilin‐2 OS=Homo sapiens 0.484 0.409

O60506 Heterogeneous nuclear ribonucleoprotein Q 1.198 1.051

Q99623 Prohibitin‐2 OS=Homo sapiens 1.222 0.706

Q06830 Peroxiredoxin‐1 OS=Homo sapiens 1.251 1.183

P62244 40S ribosomal protein S15a OS=Homo sapiens 1.265 1.124

P07355 Annexin A2 OS=Homo sapiens 1.299 1.194

P28072 Proteasome subunit beta type‐6 OS=Homo sapiens 1.302 1.133

P21333 Filamin‐A OS=Homo sapiens 1.310 1.243

Pros and Cons to iTRAQ

Expensive

Special data analysis platform

Samples come from cell cultures

Time consuming

One experiment can determine fold change, protein ID and Post translational modification

Up to several groups can be compared at the same time

Mixing at the beginning, allow more sample preparation steps

significant reduction of experiment error

High reproducibility

doesn’t use a stable isotope containing compound tochemically label the protein. It can determine the relativeamount of proteins in two or more biological samples bycomparing peptide peak areas or spectral counting.

Label-free quantification

Peak AreaMASCOT

ProteomeDiscoverer

Spectral CountingMASCOTScaffold

ProteomeDiscoverer

Relative Concentration (emPAI)MASCOT

Label Free Quantitation by Spectral CountingRelative protein quantification is achieved by comparing the number ofidentified MS/MS spectra from the same protein in each of multipleLC/MS/MS

Hongbin Liu, Rovshan G. Sadygov and John R. Yates, III, A Model for Random Sampling and Estimation of Relative Protein Abundance in Shotgun Proteomics Anal. Chem. 2004, 76, 4193-4201

# of reports = # of Treatment Conditions X # of Bio-replicates

Scaffold

Label Free Quantitation by Spectral Counting

Spectral Counting Results-Scaffold

Conditions

Bio-Replicates

P-Value

# of Spectra



Spectral Counting Results

emPAI = 10PAI -1 Protein content (mol %) = emPAI/ ∑ (emPAI) x 100

Protein name Conc.

Fmol

emPAI

Protein Content RankConc. emPAI Conc. emPA

IElongation factor 1-a 1 870 9.00 15.04 16.18 2 1a enolase 596 6.50 10.30 11.69 3 2Heat shock protein HSP 90-a 940 6.26 16.25 11.26 1 3Vimentin 336 5.58 5.81 10.03 6 414-3-3 protein 381 4.62 6.58 8.31 5 540 S ribosomal protein S16 456 2.98 7.88 5.36 4 6Pyruvate kinase, M2 isozyme 216 2.06 3.73 3.70 9 740 S ribosomal protein S9 135 1.89 2.33 3.40 12 8GTP-binding nuclear protein RAN 255 1.85 4.41 3.33 8 9ADP, ATP carrier protein, fibroblast isoform

264 1.64 4.56 2.95 7 10

Peripherin 84 1.48 1.45 2.66 18 11Stress-70 protein, mitochondrial precursor

195 1.42 3.37 2.55 11 12

Fructose-bisphosphate aldolase A 210 1.15 3.63 2.07 10 13IgE-binding protein 122 1.15 2.11 2.07 13 14Calreticulin precursor 114 1.15 1.97 2.07 14 1560 S ribosomal protein L11 108 1.15 1.87 2.07 15 1660 S ribosomal protein L17 90 1.00 1.56 1.80 17 17Peroxiredoxin 4 72 0.85 1.24 1.53 20 18Voltage-dependent anion-selective channel protein

54 0.85 0.93 1.53 21 19

T-complex protein 1, e subunit 96 0.81 1.66 1.46 16 20ATP synthase oligomycin sensitivity conferral protein

78 0.70 1.35 1.26 19 21

Phosphate carrier protein, mitochondrial precursor

48 0.55 0.83 0.99 22 22

T-complex protein 1, a subunit B 36 0.52 0.62 0.94 23 23Nucleolar RNA helicase II 30 0.45 0.52 0.81 24 24

Relative Quantitation Using emPAI

Ishihama Y1, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.Mol Cell Proteomics. 2005 Sep;4(9):1265-72

Relative Quantitation of PTM Using Peak Area

RT: 19.96 - 50.19 SM: 7B

20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50Time (min)

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RT: 31.46AA: 6948813009

RT: 36.22AA: 12379095

RT: 33.66AA: 3238508

RT: 41.54AA: 3445463

RT: 44.32AA: 3944626

RT: 48.01AA: 2336711

RT: 39.67AA: 66098364

RT: 42.77AA: 41752420

RT: 38.84AA: 31864608

RT: 33.82AA: 596224

RT: 35.79AA: 304562

RT: 31.35AA: 126488

RT: 45.02AA: 90790

RT: 49.46AA: 72789

NL: 1.75E8Base Peak m/z= 514.7983-514.8189 F: FTMS + c ESI Full ms [350.00-2000.00] MS ICIS 30138_TRY

NL: 2.39E6Base Peak m/z= 535.8032-535.8246 F: FTMS + c ESI Full ms [350.00-2000.00] MS ICIS 30138_TRY

Sequence m/z Mass Error

Peak Area RT (min)

Total Peak Area

Percentage (%)Theoretical Observed

115GELLEAIKR123 514.80862+ 514.80632+ ‐4.47 6948813009 31.46 99.40

115GELLEAIK(Ace)R123 535.81392+ 535.81362+ ‐0.56 41739255 42.77 6.99E+09 0.60

115GELLEAIKR123

115GELLEAIK(Ac)R123

(Based on Retention Time and SIC)

MALDI Imaging

Image from 2014 ASMS Workshop

MALDI Imaging

Laser Capture Microdissection Proteomics

50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 21Time (min)

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80172.3651.09 74.43

147.2873.4659.81

83.0067.5146.9484.43

125.6190.98 98.24102.59 154.41135.68

107.81 113.21 129.55 156.09140.88 161.85121.24 203.90186.96 193.16175.36

96.3074.03

72.89 97.42 164.8778.17 82.2072.6559.3690.1855.88 185.19123.9166.13 128.35

101.89153.68113.47 188.09 199.8052.73

50.86 130.21 163.13141.80 195.09178.09 208.48120.3046.39

146.51 171.8573.23 171.2978.49

97.0378.6667.29

56.18 59.48 82.84 85.96 128.6398.06 185.93113.7690.37168.18124.41 189.02 200.9953.11 153.97105.4151.41

124.14 135.09 155.6747.08 210.16184.39 200.06120.75

�Thermo LTQ Orbitrap�Bruker ultrafleXtreme MALDI-TOF TOF�Thermo Scientific LTQ�Bruker maXis UHR QTOF�Bruker amaZon ion trap with ETD�Ettan Spot Handling Workstation�Thermo Trace GC-MS�Thermo DSQ II GC-MS�Micromass Q-TOF II�FPLC System

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Protein Mass Spectrometry (Proteomics) - Ohio … · Protein Mass Spectrometry (Proteomics) ... –- DIGE ˜ Non Gel-Based ... requires clean sample Measure peptides fast experiment,