Fundamentals of Biological Mass

Spectrometry and Proteomics

Steve Carr
Broad Institute of MIT and Harvard
 Modern Mass Spectrometer (MS) Systems

Orbitrap Q-Exactive Triple Quadrupole

Discovery/Global Experiments Targeted MS

MS systems used for proteomics have 4 tasks:

•  Create ions from analyte molecules
•  Separate the ions based on charge and mass
•  Detect ions and determine their mass-to-charge
•  Select and fragment ions of interest to provide
structural information (MS/MS)
Electrospray MS: ease of coupling to liquid-based separation
methods has made it the key technology in proteomics
Possible Sample Inlets
Syringe Pump
Sample Injection
Loop
Liquid
Autosampler, HPLC
Capillary
Electrophoresis
Expansion of the
Ion Formation and
Sampling Regions
Nitrogen Drying Gas
Electrospray Atmosphere Vacuum
Needle
3-5 kV

Liquid

Nebulizing Gas
Droplets Ions
Containing
Solvated Ions
Isotopes

Most elements have more than one stable isotope.

For example, most carbon atoms have a mass of 12 Da, but in
nature, 1.1% of C atoms have an extra neutron, making their
mass 13 Da.

Why do we care?
Mass spectrometers “see” the isotope peaks provided the
resolution is high enough.
If an MS instrument has resolution high enough to resolve
these isotopes, better mass accuracy is achieved.
Stable isotopes of most abundant elements of peptides

Element Mass Abundance

H 1.0078 99.985%
2.0141 0.015
C 12.0000 98.89
13.0034 1.11
N 14.0031 99.64
15.0001 0.36
O 15.9949 99.76
16.9991 0.04
17.9992 0.20
Monoisotopic mass and isotopes
We use instruments that resolve the isotopes enabling us to accurately
measure the monoisotopic mass

Monoisotopic
Monoisotopic no 13C atoms
mass; all 12C,mass
corresponds to
One mass
lowest
13 C atom
peak

Two 13C atoms

Angiotensin I (MW = 1295.6)
(M+H)+ = C62 H90 N17 O14

TheWhen the isotopes

monoisotopic mass of aare clearly
molecule is theresolved
sum of thethe monoisotopic
accurate masses for the mass
most
abundant isotope of each element present. As the number of atoms of any given element
is used
increases, theas it is theofmost
percentage accurate
the population measurement.
of molecules having one or more atoms of a
heavier isotope of this element also increases. The most significant contributors to the
isotopic peak pattern for peptides is the 13C isotope of carbon (1.1%) and 15N peak of
nitrogen (0.36%).
Example of electrospray mass spectrum of mixture of 3 peptides

Isotope spacing = 1.0:

Ion is singly charged: (M+H)1+
MW = 584.2

Isotope spacing = 0.5: Isotope spacing = 0.3:

Doubly charged: (M+2H)2+ Triply charged: (M+3H)3+
MW = 1096.2 MW = 1806.6

586.2

603.5
Example of electrospray mass spectrum of mixture of 3 peptides

Peptide 3: MW = 1806.6 F-G-G-F-T-G-A-R-K-S-A-R-K-L-A-N-Q

Peptide 2: MW = 1096.2 F-G-G-F-T-G-A-R-K-S-A
Peptide 1: MW = 584.2 F-G-G-F-T-G
Peptide 1
(M+H)1+

Nociceptin 1-6
Peptide 2
Peptide 3
(M +2H)2+
(M+3H)3+
Nociceptin 1-11

Nociceptin 1-17
Peptide 2 586.2
(-H, +Na)
603.5
How we sequence peptides: MS/MS intact peptide parent ions

Scan m/z Pass Pass

350-1200 All ions All ions

MS

Collision
MS 1 Cell (off) MS 2 MS or mass spectrum
HPLC
Column

MS/MS
Collision
Cell (on)
Pass m/z Fragment Pass MS/MS spectrum of doubly
834-838 all ions All ions charge ion at m/z 836.5

MS/MS means using two mass analyzers (combined in one

instrument) to select an analyte (ion) from a mixture, then
generate fragments from it to give structural information.
Dominant fragment ions observed by collision-
induced dissociation (CID) of peptides

b ions y ions

Direct cleavage Rearrangement of

of peptide bond mobile proton
 Example electrospray MS/MS spectrum of a peptide

F-I-I-V-G-Y-V-D-D-T-Q-F-V-R

1199.53
100

90

80
880.46
70 Δ=115 Δ=115
Relative Abundance, %

979.49
Δ=128 Asp Asp
60 Δ=99 Δ=57 Δ=99
Gln Val
50 792.35 Gly Val
Δ=147 693.26
Phe 1142.53 1298.60
40 706.62
Δ=101 1497.46
Thr Δ=163
30
Tyr
650.44 765.40 1251.46
20 421.33
473.15 549.46 907.26 1124.44 1398.48
10 261.30 818.64

0
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 16

m/z
 Example electrospray MS/MS spectrum of a peptide

11 10 9 8 7 6 5 4 3 2 y ions

F-I-I-V-G-Y-V-D-D-T-Q-F-V-R
b ions 2 3 4 6 7 8 9 10 11 12 13

1199.53
100
Δ=115 y10
90
Asp y7
80 Δ=115 y8
Asp 880.46 Δ=99
70 Δ=57 Val
Relative Abundance, %

979.49
b7 Gly
60 Δ=128 b6 Δ=99
Gln Δ=163 y9 y11
50 b3 792.35 Val b13
Δ=101 693.26 Tyr
b4 1142.53 1298.60
40 Thr 706.62
y5 y6 b10 b11
1497.46

y2 y3
30
y4 b8
650.44 1251.46 b12
20 b2 765.40
421.33
473.15 549.46 907.26
b9
1124.44 1398.48
10 261.30 818.64

0
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 16

m/z
Discovery Proteomics: differential expression profiling by MS

Biological Samples LC-MS/MS Data Analysis

(case vs. control)

Protein Mixtures
• Biofluids Separate and Analyze Search DB using peptide
• Tissue lysates Peptides by LC-MS/MS m/z and sequence

•  digest to peptides •  m/z and intensity of peptides •  Peptide identity

•  fractionate peptides •  rich pattern •  Protein identity
•  Fragment ions for sequence •  Relative abundance
Most analyses of proteins are done by digestion
of proteins to peptides (“bottom-up” proteomics)

Advantages:
•  Data acquisition easily automated
•  Fragmentation of tryptic peptides well understood
•  Reliable software available for analysis
•  Separation of peptides to create less complex subsets
of the proteome for MS analysis is far easier than for
proteins (relates to breadth and depth of coverage)

Disadvantages:
•  Simple relationship between peptide and protein lost
•  Took highly complex mixture and made it 20-100x
more complex
•  Puts high analytical demands on instrumentation
Obtaining sequence information on
intact proteins: “top-down” proteomics
Ÿ  Most useful for single proteins or relatively simple mixtures (1)
Ÿ  Can distinguish sequence variants
Ÿ  Enables deciphering of combinatorial modification “codes” on
proteins like histones (2).

Ÿ  While useful, its not suitable for most biomedical applications
yet:
–  requires highly specialized instrumentation
–  cannot be easily applied to complex biological samples
–  Data interpretation is far more difficult and less automated
–  Breadth and depth of coverage of the proteome is orders of
magnitude less than for bottom-up proteomics

1.  Tran et al.Nature, 2011, 480(7376) p. 254-8

2.  Tian et al. Genome Biology 2012, 13:R86
A selective look into the proteomic “tool chest”

Reduction and Alkylation

Ÿ  Routinely done prior to enzymatic digestion to break disulfide
bonds, unfolding proteins to make them more susceptible to
enzymatic cleavage
A selective look into the proteomic “tool chest”

Highly Specific Proteases

Ÿ  Trypsin C-terminal to Arg and Lys
Ÿ  Lys-C C-terminal to Lys
Ÿ  Staph. V8 C-terminal to Glu and Asp
Ÿ  Asp-N N-terminal to Asp

Non-Specific Proteases
Ÿ  Chymotrypsin C-terminal to aromatic, aliphatic
(e.g., Tyr, Trp, Phe, Leu)
Ÿ  Proteinase K, Thermolysis C-terminal to aromatic, aliphatic
Discovery Proteomics: differential expression profiling by MS

Biological Samples LC-MS/MS Data Analysis

(case vs. control)

Protein Mixtures
• Biofluids Separate and Analyze Search DB using peptide
• Tissue lysates Peptides by LC-MS/MS m/z and sequence

•  digest to peptides •  m/z and intensity of peptides •  Peptide identity

•  fractionate peptides •  rich pattern •  Protein identity
•  Fragment ions for sequence •  Relative abundance
Peptide Sequencing by LC/MS/MS

peptide from MS-1 Collision MS-2

Q
COO Cell
protein of interest NH
I
E
F
K L H
2
NH2 Y
NH2 Y
G
1D or 2D LC NH2
Y G G
separation Y
G
NH2 Y
G G F
COOH
G
NH2 Y G G F L
L NH2
F COOH G G F L COOH

G F L COOH
F L
COOH
S
M A P L
A COOH
A R
Trypsin digest N S P
N

Reduce, alkylate

Select mass from Sequence

peptide of interest peptide of interest

proteins
 Automated Peptide Sequencing by LC/MS/MS
R T : 0 .0 1  -­‐  9 0 .15
Total Ion Current Trace produced during LC-MS/MS
AB
340 50 5 NL:
10 0 12 54
9 .54 E 7
510
Relative Abundance

80 385 B ase  P eak  F :  +  

465 c  F ull  ms  [  
60 1150 12 9 9 3 0 0 .0 0  -­‐  
335 4 15 645 695 10 70 114 5 2 0 0 0 .0 0 ]
40 53 5 10 15 10 6 0
295 555 6 3 0 70 5 116 5 1175
175 9 55 13 0 4 13 59
18 0 2 50 6 50 79 5 8 6 0 8 75 9 2 5
20 58 5 76 0 9 70 13 0 9
41 31 14 0 170 13 6 9 14 6 9
0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
T ime  ( min)

500.6 684.6
10 0
535.8
A
10 0
B

Relative Abundance
Relative Abundance

403.9 750.0
50 493.6 50

616.6 713.0
0 0
50 0 10 0 0 150 0 2000 50 0 10 0 0 150 0 2000
m/z m/z

6 4 4 .0

MS/MS 535.8
10 0
MS/MS 500.6

Relative Abundance
6 58 .9
10 0
Relative Abundance

6 53 .0
50 59 8 .5
52 6 .9
MS/MS 403.9 10 0
MS/MS 684.6

Relative Abundance
50 10 0
Relative Abundance

59 6 .7
511.0 2 13 .1 4 6 9 .5 56 5.1 70 0 .5 10 14 .2 1119 .3
3 18 .2 0
8 14 .7 8 4 9 .5
59 61154
.2 .6 13 6 1.6 4 71.0 6 9 8 .2 712 .6
2 0 050
50
MS/MS 493.6 10
400 0 600 8 0809 7.4 10 0 0 12 0 0
MS/MS 750.0
14 0 0

Relative Abundance
0 10 0
Relative Abundance

50 0 10 0 0 150 0 m/z
156 .9 4 9 1.5 6 9 8 .5 9 6 8 .4 9119
6 7.3
6 .5 16 0 5.5
3 74 .4 m/z 6 8 3 .2 79 6 .3 8 9 7.3
0 572 .5 659
4 1.1
6 .2 0 50 59 8 .6 12 2 5.4
50 10 04 57.3 9 4 2 .4
MS/MS 616.6 MS/MS 713.0
Relative Abundance
10 0 54 3 .7
Relative Abundance

200 440403 .1 54 36.500 800 10 0 0 12 0 0 50 0 10 0 0 10 9 5.3 150 0 2000

2 6 2 .2 m/z 755.3
2 8 9 .1 m/z 6 77.1
6 0 0778
.7 .9 8 9 2 .1 1153 .0 12 8 6 .3
0 0 4 71.0
50
50 6 77.1
1-2 sec 50 0
52 0 .0
4 8 9 .3
m/z
10 0 0
8 14 .6
8 9 3 .6 10 9 0 .2
0
50 0 10 0 0 9 54 .4
m/z
150 0
119 6 .4
2000

cycle time
0
50 0 10 0 0 150 0 2000
50 0 10 0 0 150 0
m/z
m/z

“Top 4 Method” (modern MS systems can do up to “top 20”)

MS/MS Search Engines: looking up the
answer in the back of the book

Acquired MS/MS Sequence Database

spectrum (translation of transcriptome)

Theoretical spectrum
ISLLDAQSAPLR
VVEELCPTPEGK
correlate DLLLQWCWENGK
ECDVVSNTIIAEK
GDAVFVIDALNR
VPTPNVSVVDLTNR
200 400 600 800 1000 1200 200 400 600 800 10001200
m/z m/z SYLFCMENSAEK
PEQSDLRSWTAK
similarity score
Best matching database peptide
Determine peptide FDR by searching reversed DB

Algorithms: Mascot, MaxQuant, SpectrumMill, X-Tandem…

Rolling peptides up to the protein level

Peptide 1
Peptide 2 Prot A
Peptide 3
Prot B
Peptide 4
Peptide 5
Peptide 6
Prot E
Peptide 7
Peptide 8 Prot D Prot X
Peptide 9
Peptide10 Prot Y
Prot Z

Slide courtesy of Alexey Nesvizhskii

Examples of a Protein Centric Table (MaxQuant)

Unique Sequence Mol.

Protein Gene Peptides Coverage Weight Ratio H/ Ratio H/
IDs Names Rep01 [%] [kDa] L L Count p-value

A0ELI5 Edc3 3 10.2 55.9 1.2 4 0.96

mCG_96
A0MNP4 84 1 3 33.7 1.0 1 0.01

A1A549 Tcf3 3 5.2 64.0 1.0 5 0.03

Pep$de   Ra$o  H/L  
2510012
APEPTIDEK   1.0  
A1L013 J08Rik 5 7.8 90.6 0.78 8 0.54
mCG_20 YKPSTELLIR   1.2  
A1L329 206 9 10.9 109.9 0.86 16 0.78
EWERTHEFAASLR   1.6  
mCG_19
A1L3B6 432 8 37.4 28.9 0.73 19 IAMAPEPTIDER  
0.66 0.9  
GWQIMNCSTYK   0.5  

Protein X
YHTLSSVTYEHLK   1.5  

§  Table of values organized around proteins ISEEALARGEPEPTIDEK   1.2  

Median   1.2  

§  A ratio that indicates a fold-change vs. a control condition

§  We generate a false discovery rate or p-value statistic for each protein ratio
to indicate how different from the null hypothesis (unchanged)

§  A prioritized list of candidates for follow-up studies

 Protein Inference Problem
Shared peptides: map to more than a single entry
in protein database

Prot A protein A or protein B ??

Peptide Or both?

Prot B In bottom-up proteomics the

connectivity between peptides
and proteins is lost

Shared peptides are more prevalent with databases

of higher eukaryotes due to the presence of:
§  related protein family members
§  alternative splice forms
§  partial sequences
Slide courtesy of Alexey Nesvizhskii
Peptide Quant to Protein Quant

Pep$de   Log2  SILAC  Ra$o  

§  A peptide could belong to more
APEPTIDEK   0.12   than one protein
YKPSTELLIR   0.15  
Protein  X  

EWERTHEFAASLR   0.07  
§  Go with preponderance of the
IAMAPEPTIDER   0.21  
GWQIMNCSTYK   0.14  
evidence to assign peptide
YHTLSSVTYEHLK   0.29   •  Occam’s razor principle
ISEEALARGEPEPTIDEK   0.23  

EITHERWAYK   0.22  
§  In this case, peptide is assigned
to Protein X because there are
more peptides supporting it
Protein  Y  

SIMPLESEQK   0.77  
LITTLEPEPTIDER   0.99  
Quantitative Data Drives Modern Proteomics

•  Analyze biological replicates of state comparisons

•  The end result is always a ratio:
–  WT expression vs. mutant Non-­‐specific    
–  Drug vs. no drug

 Proteins    
Specific,  

Unique  
–  Bait vs. control
upregulated  

replicate 2 (+drug/-drug)
• T-­‐staHsHcs  or  Gaussian  
Modeling  drives  calling  of  
regulaHon  or  specificity    

replicate 1 (+drug/-drug) Unique    

Proteins    
 Relative Quantification Methods for Discovery Proteomics
Label-free quantification Chemical labeling Metabolic labeling (SILAC)
(1 sample at a time) (up to 10 samples at a time) (up to 3 samples at a time)
State A State B State A State B State A State B
(light) (heavy)

Label

Combine

Label

Combine

Quantify Identify

m/z m/z m/z

• Need multiple replicates • Compression (fractionate) • Limited plex-level

• Less precise at low abund. Increasing precision
• Cost • Humans can’t be labeled
Label-free quantification: spectral counting or peak area

Label-free quantification •  One spectrum with peptide ID that can be

linked to a protein = 1 count for that protein
State A State B –  Basis of spectral counting

•  Detection likelihood is tied to abundance

–  Results vary depending on Instrument settings
and number of peptides in protein

•  Only reliable for moderate to highly abundant

proteins
–  Lots of missing data, especially for lower
abundance proteins
–  Poor precision leads to high FDR

•  Low throughput
–  Every sample run separately
–  Triplicate analyses required for stat. confidence
m/z
–  Instrument time = $; not inexpensive!
SILAC: Stable Isotope Labeling by Amino acids in Cell culture

Pros Cons
Metabolic labeling (SILAC)
(up to 3 samples at a time) Deep, highly precise quant. Limited plex level (3 max)
State A State B Works well in most cell lines Not practical for most model
(light) (heavy) systems
Works with all PTMs Can’t label humans
Label
Relatively inexpensive

Combine

State A State B

6-7 doublings in
media depleted of
light (12C6)lysine

m/z
 •  Time course of activation
•  Mixing samples improves data
and saves instrument time
•  ID of p-sites requires MS/MS
•  Detects some proteins associated
with pY-proteins
Blagoev, Ong et al. (2004) Nature Biotech. 22: 1139
Chemical Labeling of Peptides:
Multiplexed Quantification
with Isobaric Mass Tag Reagents

Mass = 145
Chemical Labeling of Peptides:
Multiplexed Quantification
with Isobaric Mass Tag Reagents

100 116.1111

80
114.1108
291.2149
Mix Peptides from all 4 Samples: analyze by MS
117.1145
9060 390.2832

8040
MS
Relative Abundance

7020
60 0
same peptide
50
112 114 116 118
m/z 720.4188 from 4 different
40 reporter ions 503.3672
MS/MS samples:
30
404.3024
703.2882 Observed
20 116.1111 218.0594 614.2397 792.3369

10
200.1014
145.1086 240.1341 331.1429 462.1813
774.3190833.5016
precursor
352.1475 549.2076 904.5338
0
561.3007 intensity = Σ of all
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
m/z labeled versions
m/z
Sequence informative fragment ions
 116.1111
100
114.1108

iTRAQ Experimental Example 80

Relative Abundance
60
Kinase Kinase Kinase
DMSO Peptide #1:
Inhib 1 Inhib 2 Inhib 3
No effect 40

20
Lyse and
0
Digest 112 114 116 118
m/z

Label 100
114.1107

“114” “115” “116” “117”

80

Relative Abundance
Pool
60
Peptide #2:
Phosphopeptide Sensitive to 40
115.1077

Enrichment all 117.1146

inhibitors 20

0
112 114 116 118
LCMS m/z

116.1117
100
114.1112
80

Relative Abundance
Peptide #3: 60

Sensitive to
40
inhibitors 1 &
117.1146
3 20

0
112 114 116 118
m/z
Isobaric tag reagents with higher multiplex levels now available: increased
sample throughput with high sensitivity and good quantitative fidelity

3x increased throughput Highly consistent quantification results

9 tumor samples (4 basal; 4 luminal; 1 reference)
ref
iTRAQ4

TMT6

TMT10

Log2 basal/luminal tumors

Analytical challenges of proteomics differ in important
ways from transcriptional analysis

Transcriptional Profiling MS-based Proteomics

Lysozyme #1047-1064 RT: 14.18-14.47 AV: 18 NL: 5.58E3
T: FTMS + p NSI Full ms2 1431.40@35.00 [ 390.00-2000.00]
993.0279
100
95
1584.7555
90
85
1167.6802
80
1408.6818
75

Relative Abundance
1352.9105
70
1638.7799
65 1538.7399
60 1839.5064
975.5341
55
50
45 1081.7426

40
35 1773.8836 1894.6001

30
25
20 1285.7981
15 660.3698 1963.1184
559.8581 806.9863
10 915.3887
497.6919
5
0
400 600 800 1000 1200 1400 1600 1800 2000
m/z

Ÿ All possible features known Ÿ All possible features not known

Ÿ Sample is static during analysis
Ÿ All features measured
Ÿ Robust means to amplify low
numbers DNA or RNA (PCR)
Ÿ Signal not detected means
feature not present
Ÿ Sample is dynamic during analysis
Ÿ 20-50% of features measured
Ÿ No protein PCR (analytics have to
deal with enormous dynamic range)
Ÿ Signal not detected means either
that feature not present or feature
present but not detected
Discovery defines a reduced set of “sentinel” marks that
need to be repeatedly measured in a range perturbations

Discovery: Assays:
• Disease Currently: Westerns, • Highly specific
• Development immunoassays • Sensitive
• Drug Future: Targeted MS, • Highly precise
• KO/KI ImmunoMS • Multiplexed
• Interference-free

Not all proteins Precisely measure

and (especially) selected analytes
PTMs observed in in all experiments
all experiments – no missing data
How do you start a proteomics project?

We meet to discuss your project

(scarr@broadinstitute.org)
•  Project proposals are reviewed for scientific merit, technical
feasibility and alignment with our interests and the Broad mission
•  Discussion of the science and experimental design
•  Sample preparation discussed in detail - what, how and by
whom
•  All projects are collaborative

Funding:
•  Platforms are largely self-supporting and must charge the work
performed. If projects are reviewed favorably but lack funding,
we will help investigators explore options for support, including
consideration for collaborative funding through the Broad.
www.broadins$tute.org/proteomics  
The Broad Institute Proteomics Group
Suggested additional reading

Carr and Annan, 2001. Overview of Peptide and Protein Analysis by

Mass Spectrometry. Current Protocols in Molecular Biology 10:
10.21.1–10.21.27.

Aebersold, R. and Mann, M. 2003. Mass spectrometry-based

proteomics. Nature 422:198-207.

Cravatt et al. 2007. The biological impact of mass-spectrometry-based

proteomics. Nature 450: 991-1000.
Additional resources for MS data interpretation

•  Manual  de  novo  tutorials    

•  Don  Hunt  and  Jeff  Shabanowitz    
•  h]p://www.ionsource.com/tutorial/DeNovo/DeNovoTOC.htm  
•  Rich  Johnson  
•  h]p://www.abrf.org/ResearchGroups/MassSpectrometry/EPosters/ms97quiz/SequencingTutorial.html  
•  Automated  de  novo  -­‐  PEAKS  
•  h]p://www.bioinformaHcssoluHons.com/peaks/tutorials/denovo.html  
•  h]p://www.youtube.com/watch?v=lyhpRu6s7Ro  
•  De  Novo  Sequencing  and  Homology  Searching  Tutorial  
•  Ma  B,  Johnson  R.  Mol  Cell  Proteomics  11: O111.014902,  1–16,  2012..  
•  ModificaHon  Site  LocalizaHon  Scoring:  Strategies  and  Performance  -­‐  Review  
•  Chalkley,  RJ  and  Clauser,  KR.  Mol  Cell  Proteomics  11,  3-­‐14,  2012  .  
•  Target/Decoy  FDR  -­‐  Tutorial  
•  Elias  &  Gygi,    Nature  Methods,  4,  207-­‐214,  2007.  
•  Protein  Inference    -­‐  Tutorial  
•  Nesvizhskii,    Mol  Cell  Proteomics,  4,  1419-­‐1440,  2005.  

41
BACKUPS  
 14+ 23,984 +/- 2
13+ Example of electrospray mass spectrum
ESI MS MALDI MS (M+H)1+ = 24,010 +/- 30
24,024 +/- 2
of intact protein (beta-Casein)

+
18+ 15

16+
Relative Abundance, %

24,093 +/- 2
12+

1400 1410 1420

11+

10+

9+
8+

800 1200 1600 2000 20000 22000 24000 26000

m/z m/z
47. M. Mann, C. K. Meng and J. B. Fenn,
Anal . Chem. 61, 1702 (1989).