Introduction to Mass spectrometry

Based Proteomics Workflow

What is proteomics?
Proteomics includes not only the identification
and quantification of proteins, but also the
determination of their localization, modifications,
interactions, activities, and, ultimately, their
function.
-Stan Fields in Science, 2001.

kD

st
d

How to think about mass spectrometry

based proteomics
Watermelon

220160120100908070-

Honeydew
Cantaloupe
Grapefruit
Pear
Apple

60-

Orange

50-

Lemon

40-

Lime
Grape

3025201510-

Cranberry
Blueberry

Dynamic Range
=103

How to think about mass spectrometry

based proteomics
mRNA

Protein

Able to amplify (PCR)

No amplification
High variability in amount (>109)

Mass Spectrometry based proteomics:

What it is and what it isnt
What it is:
A highly powerful tool for protein
identification and quantification
Complementary to other technologies and
analysis methods
What it is not:
Magic
Able to give all the answers
Simple (relatively speaking)
Cheap

What can mass spectrometry tell me?

proteins in mixtures
quantitative analysis of protein expression
post-translational modifications:
Phosphorylation

protein interactions

Mass Spectrometry Primer

A mass spectrometer
measures mass to charge ratio
or m/z
Ionization

Mass
Separation

Ion collection

MALDI
Electrospray

quadrupole
ion trap
time-of-flight

mass analysis

What do we put in our mass

spectrometer to measure m/z?
Trypsin

Protein, MW = 10,000 +

N-THK.NCPHIVVGTPGR.IPD-C
digest into peptides

Peptides,
MW < 4,000

R+-COOH

+ NH2
+ NH2
+ NH2

cleaves C-teminal side of

arginine (R) and lysine (K)

K-COOH
+

K-COOH
+

Electrospray ionization (ESI)

High voltage placed
on a fused silica
column causes a
spray of charged
droplets which
evaporate leaving
charged peptides

+NH2

NewObjective

K-COOH
+
UniversityofBristol

Matrix-assisted laser desorption/ionization

(MALDI)
CH C CN
COOH

matrix

sample

OH

UV laser

3 mm
A laser pulse excites the matrix material at its
resonant frequency and energy is imparted to
peptides, charging them.

Triple Quadrupole Mass Spectrometer

Q1

Q2

Q3

microcapillary

electrospray source
Ionization

collision cell
Mass Selection

Detector
Ion Collection

Quadrupole Optics

In a quadrupole mass spectrometer four

(quad) parallel rods (poles) are arranged
equidistantly from a central (imaginary) axis.
Charged ions are injected along the central
axis of the quadrupole assembly.
Static and alternating (radio frequency)
electric potentials are applied to opposite
pairs of rods, creating a fluctuating electric
field.

m/z

Mass Spectrometers identify peptides by

fragmenting along the amide backbone

N
N
N

N
N

Fragmentation occurs along the backbone, revisited

+NSGDIVNLGSIAGR+
b

+N
SGDIVNLGSIAGR+
+NS
GDIVNLGSIAGR+
+NSG
DIVNLGSIAGR+
+NSGD
IVNLGSIAGR+
+NSGDIV
NLGSIAGR+
+NSGDIVN
LGSIAGR+
+NSGDIVNL
GSIAGR+
+NSGDIVNLG
SIAGR+
+NSGDIVNLGS
IAGR+
+NSGDIVNLGSIA
GR+
+NSGDIVNLGSIAG
R+

Y ions
+NSGDIV

NLGSIAGR+
N L GSI AGR

bions
+NSGDIVNLGSIAGR+
yions
+N
+ NS
+ NSG
+ NSGD
+ NSGDI
+ NSGDIV
+ NSGDIVN
+ NSGDIVNL
+ NSGDIVNLG
+ NSGDIVNLGS
+ NSGDIVNLGSI
+ NSGDIVNLGSIA
+ NSGDIVNLGSIAG
+R
+ RG
+ RGA
+ RGAI
+ RGAIS
+ RGAISG
+ RGAISGL
+ RGAISGLN
+ RGAISGLNV
+ RGAISGLNVI
+ RGAISGLNVID
+ RGAISGLNVIDG
+ RGAISGLNVIDGS

bions

yions

The fragment ladder allows sequence

assignment
100

-NSGDIVNLGSIAGR-COOH

Relative Abundance

H2N

75

50

R
25

200

400

D
L

600

800

m/z

1000

1200

Quadrupole Optics cont.

The quadrupole can function in a second mode called tandem

mass spectrometry or MS/MS.
A particular peak is chosen from a MS scan and the first quad
allows only that m/z to pass into the second quad.
The second quad accelerates the species through a voltage
causing collisions with an inert gas present.
If the ion is a peptide, the collisions cause bond breakage
selectively at the amide bonds
The charged fragments enter the third quad which performs a MS
scan generating a unique pattern associated with the fragments
and thus the parent peptide
These fragments can be deconvoluted to give a peptide sequence

Tandem mass spectrometry

fragment
peptide

100

Ar

Ar

R e la tiv e A b u n d a n c e

Ar
Ar

ESI

Ar

peptides
trypsin

Re la tive Ab und anc e

gel

1. MS survey scan
100

50

600

800

1000

1200

1400

50

1200
200 400 600 8001000
80010001200

m/z

600

tandem
mass1200 spectrum
800
1000
1400

Sample Peptide
100

-NSGDIVNLGSIAGR-COOH

Relative Abundance

H2N

75

50

R
25

200

400

D
L

600

800

m/z

1000

1200

Making an identification by database

searching using SEQUEST
SEQUEST is a search program that
assigns a peptide sequence to a spectra
by comparing it to virtual spectra from a
protein database

SEQUEST Example
1. An MS/MS scan of m/z 750 and charge 2+ the
molecular weight is 1500 Da
2. SEQUEST searches a protein database starting at the
first amino acid to find all possible peptides that weight
1500 +/- 1.5
3. SEQUEST fragments each virtually and compares to the
experimental spectra.
4. For a good spectra, one peptide stands out from all
others

Scoring a match
NSGDIVNLGSIAGR

NSADIVNLGSIAGR

Acquired
spectra
Theoretical
spectra

0 1 2 3 4 5 6 7 8 9 10 11

0 1 2 3 4 5 6 7 8 9 10 11

0 1 2 3 4 5 6 7 8 9 10 11

0 1 2 3 4 5 6 7 8 9 10 11

0 1 2 3 4 5 6 7 8 9 10 11

0 1 2 3 4 5 6 7 8 9 10 11

Dotproduct

The linear ion trap LTQ

The Orbitrap and LTQ-FT

Average vs. monoisotopic mass

LTQ-FT

Time of Flight
All ions are imparted with the same energy (E) by the
application of an electric field.
E=mv2/2
m=2E/v2
Distance/time=v
m=2E/(d/t)2
Mass can be calculated based on time of arrival
because the energy, flight tube length, and time are
known.

Scanning with a ESI-tof

High mass accuracy
Low duty cycle
Large data files

ESI

Electron Transfer Dissociation

PNAS February 13, 2007 vol. 104 no. 7 21932198

ETD

Source: Thermo Fisher

ETD

ETD

Quantitative Proteomics

Why?...Comparisons
Particularly well suited to answering biologic
questions
Detail protein level changes to a biologic stimulus
Used as a tool to evaluate protein distributions
Used as a tool to follow complex formation

Modified from CEBI web site

Isotopic modification strategies

Mass spectrometers measure mass-hence
isotopically different peptides can be compared.
Single ion chromatograms for each isotope can be
compared to determine relative quantities of each.
100

light

heavy

550 560 570 580

The general formats of isotope addition

Metabolic labeling
Tagging of Cysteine
Tagging of free amines
18O labeling during digestion
Spiking in a heavy peptide to quantify a target

Isotopic tags
Label should react with a group common in
proteins
Labeling must be complete
Labeling must be stable
Label cannot inhibit ionization or fragmentation
Label must provide a large enough mass
difference for peaks to be resolved.
Light and heavy peptides should co-elute in all
separation steps preceding mass spectrometry

Stable Isotope Labeling with Amino

acids in Cell culture
SILAC
Cells are grown in media lacking
Arginine or Lysine
These amino acids are supplied as
either heavy (13C6,15N4 Arginine and
13C Lysine) or light into media
6
Serum is dialyzed to remove amino
acids

SILAC
Media w/o Lys and Arg

Heavy
Lysine,
Arginine
or both

FCS dialyzed to remove

amino acids
Unmodified
amino acids

Stimulus
of
interest

Control
Lyse cells
together

SILAC
Very simple, grow cells and lyse together.
Dialyzed serum may cause growth issues in some
cell lines
All peptides are labeled- this can challenge the
mass spec duty cycle in some experiments
Not applicable to cells that cannot be easily
propagated i.e. primary cells
Not applicable to samples where propagation is
not possible i.e. mammalian serum or organs

Protein Labeling
Isotopic tagging at the protein level is most
commonly done through cysteine or lysine
modification.
A variety of reactants are available that
incorporate thiol specific chemistry with variable
isotopic tags.
Some are selective

Amine based reagents are available as well.

Cleavable Isotope Coded Affinity Tags

Heavy reagent: 13C-ICAT

Light reagent:
12C-ICAT
O
N

N
S

S
H

Acid cleavage
O
H2N

N
N
O

Peptide

cys

Protein Quantification and Identification by the ICAT Strategy

Mixture 1

ICATlabeled
cysteines

Combine and
proteolyze
Mixture 2

Affinity
separation
with Avidin

Peptides
Proteins

ICAT
Selective and therefore reduces complexity
potentially increasing dynamic range
Most proteins have a cysteine
However:
Method involves a variety of handling steps that
can cause sample loss
Parallel cell lysis and handling can introduce
bias.

Peptide level labeling

Can be amine or cysteine based
amine based prototypes
N-isotag
iTRAQ

iTRAQ
iTRAQ (isotope Tags for Relative and Absolute
Quantitation).
Commercially available from ABI
Requires CID to generate reporter ions for
quantitation.
Requires a TOF instrument

iTRAQ schema
NH2
NH2
NH2
NH2

CID of iTRAQ labeled peptides

ITRAQ

Ross, P. L. (2004)

Copyright 2004 American Society for Biochemistry and Molecular Biology

Mol. Cell. Proteomics 3: 1154-1169

ITRAQ

Ross, P. L. (2004)

Mol. Cell. Proteomics 3: 1154-1169

Copyright 2004 American Society for Biochemistry and Molecular Biology

Labelling is consistent within one protein

Ross, P. L. (2004)

Mol. Cell. Proteomics 3: 1154-1169

Copyright 2004 American Society for Biochemistry and Molecular Biology

iTRAQ summary
Very effective method for multiplexed
quantitative studies
Trades off increased ID/quantification using
many peptides vs. selection strategy (ICAT)
Is not compatible with LC/MS strategies that
choose CID peaks based on pair intensity ratios

Label Free Quantitative

Proteomics
Isotopes are not used
Quantification is performed computationally by comparing
sequential runs
Metrics of comparison are typically AUC for aligned runs
MS/MS is either incorporated or done later as targeted
MS/MS
As an alternative, peptide counting can be used as a method
for relative quantification with MS/MS experiments.

How is this technology

implemented?

Large scale
Medium scale
Small scale

Big project example

4000 proteins
>300 orbitrap runs
Big group

Big $$$$$$

Medium Scale Proteomics

The key to medium scale proteomics is a
mechanical or affinity based preparation that
reduces the complexity of the starting material.
Examples: centrifugation, precipitation.

Peroxisome membrane
We have combined classical subcellular
fractionation with large-scale quantitative
mass spectrometry to identify proteins that
enrich specifically with peroxisomes of
Saccharomyces cerevisiae.

Micro-Proteomics
Focus on a particular protein complex
Affinity based purification
Takes advantage of existing technology
Results more approachable for an individual
investigator

Micro-Proteomics
unknown

known

Release

Identification of
binding partners

Conclusions 1
Proteomics is rapidly advancing:
Relative quantification is here.
Large scale experiments are becoming easier with
better automation tools, BUT they generate vast
amounts of data and consume significant resources.
Medium and small scale projects can be approached by
an individual investigator here and now.
Phosphorylation is observable but methodology is still
under development.

Conclusions 2
Mass spectrometers are fantastic
The results you get out are determined by what you put in.
The results you get out are determined by what you put in
The results you get out are determined by what you put in.
Problems are most often NOT the result of poor instrument
performance
Your results depend on the following:
The purity and cleanliness of you preparation
The complexity of your sample compared to the dynamic range and duty
cycle of the instrument