Protein & Peptide Analysis Linda Breci Chemistry Mass Spectrometry Facility University of Arizona MS...

Protein & Peptide Analysis

Linda BreciChemistry Mass Spectrometry Facility

University of Arizona

MS Summer Workshop

Using mass spectrometry for the measurement and/or identification of proteins

• Measuring whole proteins– Information about proteins is available on the internet– Limits due to instrument resolution, protein mass, matrix– method: MALDI/TOF – method: ESI + various analyzers

• Measuring peptides from proteins by MS– peptide mass mapping

• Gel separation steps to prepare for protein identification by MS/MS

• Identifying proteins from peptides by MS/MS

Overview

Proteins versus peptides

Enzyme

Protein Peptides

Analysis of whole proteinsGood news & bad news

• MALDI-TOF = measure with 1 or 2 protons

• ESI-Ion Trap = measure with many protons (high charge state)

• Result = mass accuracy not good enough to identify protein (but still useful!)– Mass accuracy decreases as size increases

Same protein, 2 ionization methods

MALDI/TOF – whole protein detected

10000 20000 30000 40000

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

[2M+H]+

14318.68

[M+2H]2+

7157.18

[M+H]+

14318.68

Inte

nsi

ty

m/z

ESI: Protein MW can be calculated from a protein’s charge distribution

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

0

25

50

75

100

5000 10000 15000

0

25

50

75

100

14306.0

Inte

nsi

ty

m/z

+81789.00

+91590.33

+131101.40

+101431.47

+111301.53

+121193.20

Re

lativ

e In

ten

sity

m/z

CalculatedMass Spectrum

We measure ISOTOPES (not averages)Example: Carbon is 12.000 (not 12.0107)

For every 12C there is 1.1% 13C

10 carbons

100

10.8

0.50

20

40

60

80

100

120

1 2 3 4 5 6 7

100 carbons

92.5

100

53.5

18.8

51 0.2

0

20

40

60

80

100

120

1 2 3 4 5 6 7

Peak broadening in high mass measurement

Theoretical isotope distribution for a small protein

9th Isotope14313.906

1st Isotope14304.885

Isotope # m/z % Maximum0 14304.885 0.21 14305.888 1.22 14306.891 4.63 14307.893 12.84 14308.896 26.95 14309.898 46.36 14310.900 67.67 14311.902 86.38 14312.904 97.79 14313.906 100.010 14314.908 93.511 14315.910 80.412 14316.912 64.213 14317.914 47.814 14318.916 33.415 14319.918 21.816 14320.920 13.217 14321.922 7.518 14322.924 3.919 14323.925 1.820 14324.927 0.721 14325.929 0.2


Resolution = Mass Accuracy

MASS RANGE Resolution Accuracy (Error)

m/z (at m/z 1,000) (at m/z 1,000)

2,000 (full scan)

10,000 (zoom scan)

0.006% (60 ppm) Ext. Cal.

0.003% (30ppm) Int.Cal.

INSTRUMENT

to 4,000FTICR

MALDI/TOF to 400,000

LCQ (Ion Trap)

15,000 (reflectron)

0.0001% (1ppm)

0.03% (300 ppm)to 2,000

500,000

610)(

lMWTheoretica

MeasuredMWlMWTheoreticappm


No reflectron for high masses = reduced resolution

Only multiply charged proteins observed(more peaks/mass unit)

Examples of post translational modificationsabbreviation monoisotopic average

Acetylation ACET 42.0106 42.0373Amidation AMID -0.9840 -0.9847Beta-methylthiolation BMTH 45.9877 46.0869Biotin BIOT 226.0776 226.2934Carbamylation CAM 43.0058 43.0250Citrullination CITR 0.9840 0.9848C-Mannosylation CMAN 162.0528 162.1424Deamidation DEAM 0.9840 0.9847N-acyl diglyceride cysteine (tripalmitate) DIAC 788.7258 789.3202Dimethylation DIMETH 28.0314 28.0538FAD FAD 783.1415 783.5420Farnesylation FARN 204.1878 204.3556Formylation FORM 27.9949 28.0104Geranyl-geranyl GERA 272.2504 272.4741Gamma-carboxyglutamic acid GGLU 43.9898 44.0098O-GlcNAc GLCN 203.0794 203.1950Glucosylation (Glycation) GLUC 162.0528 162.1424Hydroxylation HYDR 15.9949 15.9994Lipoyl LIPY 188.0330 188.3027Methylation METH 14.0157 14.0269Myristoylation MYRI 210.1984 210.3598Palmitoylation PALM 238.2297 238.4136Phosphorylation PHOS 79.9663 79.9799Pyridoxal phosphate PLP 229.0140 229.1290Phosphopantetheine PPAN 339.0780 339.3234Pyrrolidone carboxylic acid PYRR -17.0266 -17.0306Sulfation SULF 79.9568 80.0642Trimethylation TRIMETH 42.0471 42.0807

Mass changes are difficult to identify in high mass measurements

Computer Exercises -- Goals

• Exercise #2, Whole protein analysis– Explore Expasy information available for a protein– Find the theoretical MW of a protein– Find the amino acid sequence of a protein in FASTA format

for use in another exercise– Explore the X-ray crystal structure of a protein

Open Webpage: http://www.chem.arizona.edu/facilities/msf/BiochemLab/exercises.html

Proteins versus peptides

Enzyme

Protein Peptides

Identification of proteins from peptide analysis

Separate by 2-D (or 1-D) Gel

Remove protein from gel after cutting into peptides with an enzyme (trypsin)

We can identify hundreds of proteins in one experiment

Extracting and Separating proteins

• Extracting proteins from biological organisms– Results in complex mixture of proteins– May require detergents, etc. that complicate Mass Spec analysis– Remove contaminants (filtration, dialysis, SPE, etc.)

• Separating proteins– 1D SDS-PAGE

• Cross linking controls MW separated• Low resolution technique, spot can contain 10's to 100's of

proteins – 2D SDS-PAGE

• Best method for complex protein mixtures (IEF + SDS-PAGE)– Preparative isolectric focusing (IEF)– Reverse phase HPLC– Size exclusion chromotography– Ion exchange chromatography– Affinity chromatography

2-D Electrophoresis

• 1st Dimension: Isoelectric Focusing (IEF)– Requires maximal resolution of a

target group of proteins – Uses Immobiline DryStrip gels

(various lengths and pH gradients)

– IPGphor programmed to hydrate and separate proteins by pI (i.e. overnight)

• 2nd Dimension: Gel Separation– Apply the Immobiline DryStrip to

the top of a gel– Separation by molecular weight is

rapid (6-10 hours)

#1) pI

#2) MW

2-D Electrophoresis

• Standard Method:– Separate proteins on 2-dimensional gels– Spots (and changes) can be observed (manual or with

computer aid).– Method is reproducible (multiple runs required)– Cut out and identify spots of interest

• Gel Electrophoresis (DIGE)– Two or more samples for comparative analysis are labelled

with different fluorescent dyes, mixed together, run on the same 2D gel, and interrogated with a multi-wavelength fluorescent scanner

– Allows quantitation of subtle changes in protein expression levels between samples, without inter-gel variability - Very good for quantitation of subtle protein expression changes

– Following example: analysis of a Bordetella broncheoseptica enzyme knockout cell line, compared to wild type.

10/29/03 Gel 2: Multiplexed gel imagepH 3 pH 7

sma

ll

Mo

lecu

lar

We

igh

t

larg

e

10/29/03 Gel 2: Side-by-side Cropped Grayscale Images of WT (Cy3) and ∆dnt (Cy5)

WT (Cy3) ∆dnt (Cy5)

pH 3 pH 7 pH 3 pH 7

BB3856 – AZURINL.AAECSVDIAGTDQM#QFDK.KA.AEC*SVDIAGTDQM#QFDK.KK.QFTVNLK.HK.DGIAAGLDNQYLK.A

nanoLC-MS/MS identification of two

differentially expressed protein spots

∆dnt (Cy5)

BB3856 – AZURINK.TADMQAVEK.DK.VLGGGESDSVTFDVAK.LK.DGIAAGLDNQYLK.A

WT (Cy3)

In-Gel enzymatic digestion(trypsin most common)

Trypsin after K, RChymotrypsin after W, Y, F, before PGlu C (V8 protease) after E, D, before PLys C after K, before PAsp N after D

Proteases and Cleavage Specificities


• Exercise #3, 2-D Gel Electrophoresis– Find a gel image online containing a protein spot of interest– Explore the gel images of various organisms


Analysis of peptides from proteins

• MALDI-TOF = measure mass of peptides– peptide mass mapping

• ESI-Ion Trap = measure mass/charge of peptide– PLUS can select and fragment (MS/MS) for more information

• Result = possible to identify a protein, or identify SNP’s or modifications made to a protein

Peptide Mass Mapping using MALDI-TOF

MS of a peptide mixture by MALDI/TOF

m/z500 2500

90

0

D:\011003_500fmol\Bsaintcal\2Ref\pdata\1\1r (11:26 10/04/01)

x 4.0

Ref

Ref

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


• Exercise #4, Peptide Mass Mapping– Identify a protein from a peptide mass list– Confirm this identity by producing a theoretical mass list– Optional (for the speedy ones) identify more unknowns from

mass lists


Unknown proteins

• 66 = Bovine Serum Albumin

• 116 = beta-galactosidase from e.coli

• 55 = glutamic dehydrogenase from bovine liver

• 36 = glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle

LC/LC-MS/MS for Complex Mixtures

SCX = Strong cation exchange

RP = Reverse Phase (C-18)

Alternate an increasing salt gradient (move some peptides onto RP)

Follow by RP gradient (separate peptides, send to mass spec)

SCX RP

MS/MS

peptides from many

proteinsResults in thousands of mass spectra

A computational challenge!

MS/MS Method Using ESI

Ion Currentover 60 min

MS

MS/MS

Peptide precursor ions observed by MS

MH+

m/z = 1141.3

[M+ 2H]2+

m/z = 571.2

calculation of MH+

571.2 m/z measured x 2 1,142.4 [M+2H] - 1.0 1,141.4 [M+H]

MS-MS of 571.2

895.25

Data Analysis for MS/MS Sequencing Method

?MS/MS

MS Peptide MWFound in Selected

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

protein peptides identify

rank

200 400 600 800 1000 1200 1400 1600

Rel

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e In

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ity

m/z

theoretical spectra200 400 600 800 1000 1200 1400 1600

0

20000

40000

60000

80000

100000

120000

Re

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nte

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m/z

compare

FPhe

GGly

TThr

DAsp

MMet

DAsp

NAsn

y series ions

V F G T D M D N S R

895.25

y3

376.2y4

491.1y4

622.2

y5y6y7y8

Peptide bond fragment ions

Peptide fragment ions

H2N CH C

H

O

HN CH C

H

O

HN CH C

H

O

HN CH C

H

OH

O

CH

R

H2N C

N

CH

R'O

H

Internal immonium ion Amino acid immonium ion

a2

b2

c2

x2

y2

z2

H2N CH

R

Peptide Sequencing

mass amino acid

Alanine ALA A 71.09

Arginine ARG R 156.19

Aspartic Acid ASP D 115.09

Asparagine ASN N 114.11

Cysteine CYS C 103.15

Glutamic Acid GLU E 129.12

Glutamine GLN Q 128.14

Glycine GLY G 57.05

Histidine HIS H 137.14

Isoleucine ILE I 113.16

Leucine LEU L 113.16

Lysine LYS K 128.17

Methionine MET M 131.19

Phenylalanine PHE F 147.18

Proline PRO P 97.12

Serine SER S 87.08

Threonine THR T 101.11

Tryptophan TRP W 186.12

Tyrosine TYR Y 163.18

Valine VAL V 99.14

C

O

HN CH C

CH3

O

HN CH C

CH2

O

C

OH

O

HN

71 u. 115 u.

Ala Asp


• Exercise #5, Peptide Sequencing & Protein ID– Identify a peptide (and it’s protein) from an MS-MS mass list


Homology search to find protein functionBLAST: Computer Exercise #6

• Peptide sequences found for an “unknown” protein by Sequest database searching

• Find a possible function of this protein

LocusSpectrum Count

Sequence Coverage Length Descriptive Name

CL001145.84_fgenesh_1_aa 1 3.20% 1013 Unknown

FileName XCorr DeltCN M+H+ Sequence

lb100404_01.2750.2750.2 2.7258 0.181 3290.86RLVVVNAKPTAASAVGLAGPGAADVLPFVEADLKKS

lb100404_01.1675.1675.2 3.6422 0.181 1658.85 RHFFAAAAGQPPPQY.L


• Exercise #6, Blast Search– Perform a BLAST search for a peptide sequence that was

found in the previous exercise– Observe the other proteins with similar sequence– Not all organisms have full genomic information – homology

sequencing is useful for protein identification



• Exercise #7, Find an unknown protein– Use the same method of #4 to find an unknown peptide– Information provided:

• MS spectrum

• MS/MS spectrum


Open source software for high-throughput proteomics: X Tandem

• Current trends to free software• The Global Proteome Machine http://www.thegpm.org/

– X Tandem– Sequenced peptide libraries– Software available to programmers


• X Tandem identification of the same spectra

• Exercise #8, Find an unknown protein


Protein & Peptide Analysis Linda Breci Chemistry Mass Spectrometry Facility University of Arizona MS...

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