LC-LC-MS-MS for Complex Mixtures

Shotgun identification of protein from

protein complexes

peptides
Results in thousands of mass
from many spectra
proteins A computational challenge!

MS/MS Method Using ESI

1
 A Peptide fragmentation files

Multiple Peptide fragmentation files SEQUEST summary output example

peptides XCorr proteins

SEQUEST summary output example

Protein summary

Shotgun identification of protein

modifications from protein complexes

2
 Studying proteins: Mass spec (MS) Tandem mass spec (MS/MS)
Isolates individual peptide fragments for
(trypsin)
Mass spectrometry – method of separating (trypsin) 2nd mass spec – can obtain peptide
molecules based on mass/charge ratio sequence

eg. MALDI-TOF
Compare peptide m/z Compare peptide sequence
with protein databases with protein databases

Phosphorylation sites mapping Protein identification: 2D gel + MS

TSTEPQBQPGENL
B=phosphotyrosine b7-b6 = 243
Separate protein mixture on
MSMS OF 1543 DHB 26 (0.504) AM (Cen,2, 50.00, Ar,5000.0,0.00,0.30); Sm (Mn, 1x2.00); Cm (2:27) TOF MSMS 1543.00ES+
100
b8
1015.3774
y''9
1125.4500
4.01e3
2D SDS-PAGE

PQBQ 1126.4546
y''5
QBQP
529.2648
597.2139
1016.3760

1544.5708
997.3692

y''6
657.3303
1543.5778
PQB
BQP c9
1127.4558
469.1490 b6
1543.5295
644.29 b7
979.3594
887.3309 1017.3768

b11 1526.5491 1545.5356

658.3300 1298.4583
QP
PQ
EN~
226.1226
QBQPGEN^
869.3159
904.3628
1128.4614 y''10
1254.4733
1299.4668 b12
1412.4886

1508.5469
1546.5725

1547.5634
Excise individual spots
659.3278 809.3040 1018.3921 1300.4686

0
155.1096 187.0850
100 200
284.1253

300
398.1670

400 500 600 700 800 900 1000 1100

1170.4174

1200 1300 1400

1507.5514

1500
1548.6073
m/z for MS

2D Liquid chromatography-MS/MS Protein modifications

ƒ Examples: phosphorylation, methylation, oxidation,
(trypsin)
acetylation, glycosylation
Peptides all bind to cation
Study protein exchange column ƒ Biologically important – eg. signal transduction, regulation
complexes
without gel Successive elutions with
increasing salt gradients ƒ Cannot be deduced directly from nucleotide sequence
electrophoresis separates peptides by charge

ƒ Characterizationgenerally difficult – requires specific

Peptides are separated by antibodies or enrichment procedures
hydrophobicity on reverse
phase column
ƒ Can be identified by MS because they result in a different
Complex mixture is
peptide mass
simplified prior to
MS/MS by 2D LC
ƒ Previous
MS techniques – require enrichment and so are
more complex and modification-specific

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Identifying modifications - 2D LC-MS/MS Experiment

ƒ MS is not always accurate enough to determine modification

from a single type of peptide, especially if modification is rare Objective: Demonstrate efficacy of modification identification
using 2D LC-MS/MS
Example: phosphorylated serine AVANESGANFVK

2D LC-
Trypsin MS/MS Only single peptide Method: Do 2D LC-MS/MS to identify proteins and
AVANESGANFVK produced modifications using:
digestion
1. Defined mixture of proteins
ƒ Do multiple digestion with different enzymes Ä multiple
overlapping fragments 2. Large protein complex
ANESGANFVKNMQ Different peptides 3. Entire tissue
Trypsin, 2D LC-
MS/MS
AVANESGANFVK Ä more confidence
subtilisin, SDIPFAVANESGAN in identification of
elastase modification

Defined protein mix: Method Defined protein mix: Method

Phosphorylase A Phosphorylase B
1:10 Myosin heavy chain
ƒ One known
phosphorylation site β-galactosidase
Serum albumin
ƒ Phosphorylase B is Ovalbumin
unphos. form

trypsin subtilisin elastase

Database searches to
identify peptides (SEQUEST)

Search for modifications:

Pool peptides
Phosphorylation
Acetylation
Methylation
2D LC MS/MS Oxidation

Defined protein mix: Results Cdc2p protein complex: Method

S. pombe with TAP-tagged Cdc2p ƒ Is phosphorylated
ƒ High sequence
coverage ƒ Interacts with many
Isolate Cdc2p complex by TAP proteins (some
ƒ Overlapping
protocol (does not dissociate phosphorylated)
members of complex)
peptides at ƒ TAP-tagged
modification sites
ƒ Novel
modification sites
found in trypsin subtilisin elastase
Peptides are not
ovalbumin and
pooled
myosin
ƒ Met oxidation 3 x 2D LC MS/MS
artifactual?
Identify proteins and modifications

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 Cdc2p protein complex: Results Cdc2p protein complex: Results
ƒ >200 proteins identified
ƒ Wide range of protein concentrations
Known
ƒ 20 proteins with > 40% sequence coverage
phosphorylation
sites identified
•CONTROL OF THE EUKARYOTIC CELL CYCLE

Methylation sites
ƒ Multiple digestions increase sequence coverage
of unknown
biological
significance

Cdc2p protein complex: Results Lens tissue: Method

ƒ Phosphorylation sites in Cdc13 not resolvable Lens tissue from cataract patient ƒ Not too complex
(close proximity) ƒ High concentrations
of some proteins
•Single-stranded telomeric DNA-binding protein that (crystallins)
regulates telomere replication. ƒ Various kinds of
known modifications

trypsin subtilisin elastase ƒ Expect regulation by

modification

3 x 2D LC MS/MS

Identify proteins and modifications

Lens tissue: Results Lens tissue: Results

ƒ 270 proteins identified ƒ Known and novel
sites of
ƒ 52 with >40% sequence coverage, including crystallins phosphorylation
identified
ƒ 11 crystallins with total 73 modifications
ƒ Known oxidation
ƒ 13 of 18 known from human/bovine (different species / sites identified
time point / disease state / problem with method?) (but may be
artifact of sample
ƒ 60 novel
preparation)
ƒ Acetylations (may
be artificial
carbamylations)
ƒ Methylations not
previously
reported

5
Non-isotopic labeling

Isotopic labeling

6
 2D-PAGE IMAGE ANALYSIS DIFFERENTIAL GEL ELECTROPHORESIS (DIGE)

Cy3 Image A
Cells A
λE-Cy3
A B Gel analysis
mix 2-D PAGE Gel imaging (overlay and quantitation)

λE-Cy5
Cells B
Cy5 Image B

Excise spot; elute; digest

Extract peptides;
MS analyze MS
Protein identification IDENTIFICATION
Unlu et al. Electrophoresis 18 2071-77. (1997)

METABOLIC LABELING

λE-Cy5 λE-Cy3
mix
Cells A
extraction reduction alkylation digestion

Cells B
separation

Excise spots; elute;

digest, extract peptides;
MS analyze, Protein MS
identification IDENTIFICATION
Enriched Medium
(13C, 15N) and
QUANTITATION
Oda Y, et al. P Natl. Acad. Sci. USA 96 6591-6596 (1999)
Pasa-Tolic L, et al. J Am Chem. Soc. 121 7949-7950 (1999)

MS analysis of an E. coli proteome samples

Metabolic labeling for HPLC-MS analysis
normal vs. isotopically depleted medium
labeled
Cells

unlabeled Protein
extraction HPLC MS
Cells
+
Labeled
Cells
+IPTG

E. Coli growing in M9 medium with glucose (either

labeled or unlabeled) as the only source of carbon

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normal vs. N15-enriched medium
PROTEOLYTIC LABELING
18O water

Cells A

extraction reduction alkylation digestion

Cells B separation

MS
IDENTIFICATION
Stewart T. et al. R.ap. Comm.. Mass Spectrom. 15(24) 2456. (2001)
and
Yao XD et al. Anal. Chem.73(13):2836-2842. 2001
QUANTITATION

Enzymatic digestion CHEMICAL LABELING

R-C NH-R’ R-C NH-R’

O O Cells A
digestion
extraction reduction alkylation
enzyme H2O enzyme H2 (18)O
Cells B separation

R-C (18)OH
R-C OH
MS
O + O + H2N-R’ IDENTIFICATION
H2N-R’
and
Regnier FE et Al. J. Mass Spectrom. 37(2)133-45 (2002) QUANTITATION

CHEMICAL
REACTION
INTERFACE

The chemical reaction interface for mass spectrometry CF4

(CRIMS) C nH nO nN nS nP r SF6 NF3
HF Helium
Helium
PF5
from NF
HPLC 3 to MS

8
CRIMS SET UP ISOTOPE-CODE AFFINITY TAG (ICAT)

Helium

Desolvation Helium “heavy” ICAT

HPLC
Removal
HPLC
solvents Reactant gas “light” ICAT

Reactive Group Linker containing the

Biotin Tag
CHEMICAL Isotope Tag
MS REACTION
Gigy et al.Nat. Biotech. 17: (10) 994-999 (1999)

ICAT reagent

ICAT

ICAT strategy

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Mutiple Dimensional Liquid Chromatography Reduction of complexity
(MDLC)

Mutiple Dimensional Liquid Chromatography MS/MS

(MDLC)
Multiple charged peptides

Peptides labelled with one or two of ICAT

10
 A spectrum of ICAT-coated peptide

Sequence identification in protein mixture Regulation of ADH1 and ADH2 in yeasts

Discovery Proteomics: A Novel Approach to Quantitate ICAT procedure

Protein Expression Differences in Human Hepatocellular
Carcinoma

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ICAT strategy Profile of MDLC

Pro ICAT Pro ICAT

Pro ICAT

Software

12
Capillary Array

13
 Coupling Liquid-Phase
Separations and MALDI–MS

Principle of electrically mediated liquid deposition

On-line vacuum deposition inlet incorporating an infusio

capillary

2DE-ICAT

14
 Phosphoprotein isotope-coded affinity tag (PhIAT)
labeling method

PhIAT
1,2-ethanedithiol

15
Phosphoprotein isotope-coded affinity tag (PhIAT)
labeling method

Cleavable ICAT reagent

ICAT related litratures

16
Myc proto-oncogene protein
•Participates in the regulation of gene transcription.

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18
 Lysosomal storage diseases

Substrate conjugate for GM1 gangliosidosis

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