Mass Spectrometry:

Methods & Theory

Proteomics Tools
Molecular Biology Tools
Separation & Display Tools
Protein Identification Tools
Protein Structure Tools

Mass Spectrometry Needs

Ionization-how the protein is injected in to the
MS machine
Separation-Mass and Charge is determined
Activation-protein are broken into smaller
fragments (peptides/AAs)
Mass Determination-m/z ratios are
determined for the ionized protein
fragments/peptides

Protein Identification
2D-GE + MALDI-MS

Peptide Mass Fingerprinting (PMF)

2D-GE + MS-MS

MS Peptide Sequencing/Fragment Ion Searching

Multidimensional LC + MS-MS

ICAT Methods (isotope labelling)

MudPIT (Multidimensional Protein Ident. Tech.)

1D-GE + LC + MS-MS
De Novo Peptide Sequencing

Mass Spectrometry (MS)

Introduce sample to the instrument
Generate ions in the gas phase
Separate ions on the basis of differences in
m/z with a mass analyzer
Detect ions

How does a mass spectrometer work?

Create ions

Ionization
method

MALDI
Electrospray
(Proteins must be
charged and dry)

Separate ions
Mass analyzer
MALDI-TOF

MW

Triple Quadrapole

AA seq

MALDI-QqTOF

AA seq and MW

QqTOF

AA seq and protein modif.

Detect ions

Mass
spectrum
Database
analysis

Generalized Protein Identification by MS

Library

Spot removed
from gel

Artificial
spectra built

Fragmented
using trypsin

Spectrum of
fragments
generated

MATCH

Artificially
trypsinated

Database of
sequences
(i.e. SwissProt)

Methods for
protein
identification

MS Principles
Different elements can be uniquely
identified by their mass

MS Principles
Different compounds can be uniquely
identified by their mass
Butorphanol
N -CH2OH

L-dopa

Ethanol
COOH

HO

-CH2CH-NH2

CH3CH2OH

HO

HO

MW = 327.1

MW = 197.2

MW = 46.1

Mass Spectrometry
Analytical method to measure the
molecular or atomic weight of samples

Weighing proteins
A mass spectrometer creates charged particles (ions) from molecules.
Common way is to add or take away an ions:

NaCl + e NaCl
-

NaCl NaCl+ + eIt then analyzes those ions to provide information about the molecular
weight of the compound and its chemical structure.

Mass Spectrometry
For small organic molecules the MW can be
determined to within 5 ppm or 0.0005% which
is sufficiently accurate to confirm the
molecular formula from mass alone
For large biomolecules the MW can be
determined within an accuracy of 0.01% (i.e.
within 5 Da for a 50 kD protein)
Recall 1 dalton = 1 atomic mass unit (1 amu)

MS History
JJ Thomson built MS prototype to measure
m/z of electron, awarded Nobel Prize in 1906
MS concept first put into practice by Francis
Aston, a physicist working in Cambridge
England in 1919
Designed to measure mass of elements
Aston Awarded Nobel Prize in 1922

MS History
1948-52 - Time of Flight (TOF) mass
analyzers introduced
1955 - Quadrupole ion filters introduced by
W. Paul, also invents the ion trap in 1983
(wins 1989 Nobel Prize)
1968 - Tandem mass spectrometer appears
Mass spectrometers are now one of the
MOST POWERFUL ANALYTIC TOOLS IN
CHEMISTRY

MS Principles
Find a way to charge an atom or
molecule (ionization)
Place charged atom or molecule in a
magnetic field or subject it to an electric
field and measure its speed or radius of
curvature relative to its mass-to-charge
ratio (mass analyzer)
Detect ions using microchannel plate or
photomultiplier tube

Mass Spec Principles

Sample
+
_

Ionizer

Mass Analyzer

Detector

How does a mass spectrometer work?

Create ions

Separate ions

Ionization
method

Mass analyzer

MALDI
Electrospray
(Proteins must be
charged and dry)

Detect ions

Mass
MALDI-TOF
spectrum
MW
Database
Triple Quadrapole
AA seq
analysis

MALDI-QqTOF
AA seq and MW
QqTOF
AA seq and
protein modif.

Mass spectrometers

L in e a r T im e O f F lig h t t u b e

io n s o u r c e

Time of flight (TOF) (MALDI)

Measures the time required for ions to fly down the length
of a chamber.
Often combined with MALDI (MALDI-TOF) Detections
R e f l e c t o r T i m e O from
f F lig h t tu b e
multiple laser bursts are averaged. Multiple laser

d e te c to r

t im e o f f l ig h t

io n s o u r c e

Tandem MS- MS/MS

-separation and identification of compounds in complex
mixtures
- induce fragmentation and mass analyze the fragment ions.
- Uses two or more mass analyzers/filters separated by a
collision cell filled with Argon or Xenon
d e te c to r

Different MS-MS configurations

Quadrupole-quadrupole (low energy)

Magnetic sector-quadrupole (high)
Quadrupole-time-of-flight (low energy)
Time-of-flight-time-of-flight (low energy)

r e f le c t o r

tim e o f flig h t

Typical Mass Spectrometer

LC/LC-MS/MS-Tandem LC, Tandem MS

Typical Mass Spectrum

Characterized by sharp, narrow peaks
X-axis position indicates the m/z ratio of a
given ion (for singly charged ions this
corresponds to the mass of the ion)
Height of peak indicates the relative
abundance of a given ion (not reliable for
quantitation)
Peak intensity indicates the ions ability to
desorb or fly (some fly better than others)

All proteins are sorted based on a

mass to charge ratio (m/z)

m/z ratio:
Molecular weight divided by the
charge on this protein

Typical Mass Spectrum

Relative
Abundance

aspirin

120 m/z-for singly charged ion this is the mass

Resolution & Resolving Power

Width of peak indicates the resolution of the
MS instrument

The better the resolution or resolving power,

the better the instrument and the better the
mass accuracy
Resolving power is defined as:

M
M

M is the mass number of the observed mass

(M) is the difference between two masses
that can be separated

Resolution in MS

Resolution in MS
783.455

QTOF
784.465

785.475

783.6

Mass Spectrometer Schematic

Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps

High Vacuum System

Inlet

Sample Plate
Target
HPLC
GC
Solids probe

Ion
Source

Mass
Filter

MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI

TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS

Detector

Microch plate
Electron Mult.
Hybrid Detec.

Data
System
PCs
UNIX
Mac

Different Ionization Methods

Electron Impact (EI - Hard method)

small molecules, 1-1000 Daltons, structure

Fast Atom Bombardment (FAB Semi-hard)

peptides, sugars, up to 6000 Daltons

Electrospray Ionization (ESI - Soft)

peptides, proteins, up to 200,000 Daltons

Matrix Assisted Laser Desorption (MALDI-Soft)

peptides, proteins, DNA, up to 500 kD

Electron Impact Ionization

Sample introduced into instrument by
heating it until it evaporates
Gas phase sample is bombarded with
electrons coming from rhenium or
tungsten filament (energy = 70 eV)
Molecule is shattered into fragments (70
eV >> 5 eV bonds)
Fragments sent to mass analyzer

EI Fragmentation of CH3OH
CH3OH

CH3OH+

CH3OH

CH2O=H+

CH3OH

CH2O=H+

+ H

CH3 + OH

CHO=H+ + H

Why wouldnt Electron Impact be suitable

for analyzing proteins?

Why You Cant Use EI For

Analyzing Proteins

EI shatters chemical bonds

Any given protein contains 20 different

amino acids
EI would shatter the protein into not only
into amino acids but also amino acid subfragments and even peptides of 2,3,4
amino acids
Result is 10,000s of different signals from
a single protein -- too complex to analyze

Soft Ionization Methods

337 nm UV laser
Fluid (no salt)
+
_
cyano-hydroxy
cinnamic acid

Gold tip needle

MALDI

ESI

Soft Ionization
Soft ionization techniques keep the
molecule of interest fully intact
Electro-spray ionization first conceived in
1960s by Malcolm Dole but put into
practice in 1980s by John Fenn (Yale)
MALDI first introduced in 1985 by Franz
Hillenkamp and Michael Karas (Frankfurt)
Made it possible to analyze large
molecules via inexpensive mass analyzers
such as quadrupole, ion trap and TOF

Ionization methods

Electrospray mass spectrometry (ESI-MS)

Liquid containing analyte is forced through a steel capillary at high voltage
to electrostatically disperse analyte. Charge imparted from rapidly
evaporating liquid.

Electrospray Ionization
Sample dissolved in polar, volatile buffer
(no salts) and pumped through a stainless
steel capillary (70 - 150 m) at a rate of 10100 L/min
Strong voltage (3-4 kV) applied at tip along
with flow of nebulizing gas causes the
sample to nebulize or aerosolize
Aerosol is directed through regions of
higher vacuum until droplets evaporate to
near atomic size (still carrying charges)

Electrospray (Detail)

Electrospray Ionization
Can be modified to nanospray system
with flow < 1 L/min
Very sensitive technique, requires less
than a picomole of material
Strongly affected by salts & detergents
Positive ion mode measures (M + H)+ (add
formic acid to solvent)
Negative ion mode measures (M - H)- (add
ammonia to solvent)

Positive or Negative Ion Mode?

If the sample has functional groups that
readily accept H+ (such as amide and
amino groups found in peptides and
proteins) then positive ion detection is
used-PROTEINS
If a sample has functional groups that
readily lose a proton (such as carboxylic
acids and hydroxyls as found in nucleic
acids and sugars) then negative ion
detection is used-DNA

Matrix-Assisted Laser
Desorption Ionization
337 nm UV laser

cyano-hydroxy
cinnamic acid

MALDI

MALDI
Sample is ionized by bombarding sample
with laser light
Sample is mixed with a UV absorbant
matrix (sinapinic acid for proteins, 4hydroxycinnaminic acid for peptides)
Light wavelength matches that of
absorbance maximum of matrix so that
the matrix transfers some of its energy to
the analyte (leads to ion sputtering)

HT Spotting on a MALDI Plate

MALDI Ionization
Matrix

+
+ +-+

Laser
Analyte

+
+ ++ + --+
-+
+

+
+

Absorption of UV radiation
by chromophoric matrix and
ionization of matrix
Dissociation of matrix,
phase change to supercompressed gas, charge
transfer to analyte molecule
Expansion of matrix at
supersonic velocity, analyte
trapped in expanding matrix
plume (explosion/popping)

MALDI
Unlike ESI, MALDI generates spectra that
have just a singly charged ion
Positive mode generates ions of M + H
Negative mode generates ions of M - H
Generally more robust that ESI (tolerates
salts and nonvolatile components)
Easier to use and maintain, capable of
higher throughput

Principal for MALDI-TOF MASS

p e p tid e m ix t u r e
e m b e d d e d in
lig h t a b s o r b in g
c h e m ic a ls ( m a t r ix )

+
+ +

p u ls e d
U V o r I R la s e r
(3 -4 n s )

vacuum
+

s tro n g
e le c tr ic
fie ld

Vacc

c lo u d o f
p ro to n a te d
p e p tid e m o le c u le s

d e te c to r

T im e O f F lig h t tu b e

Principal for MALDI-TOF MASS

L in e a r T im e O f F lig h t tu b e
io n s o u r c e

d e te c to r

t im e o f flig h t

R e fle c to r T im e O f F lig h t tu b e
io n s o u r c e

d e te c to r

r e f le c t o r

t im e o f flig h t

MALDI = SELDI
337 nm UV laser

cyano-hydroxy
cinnaminic acid

MALDI

MALDI/SELDI Spectra
Normal

Tumor

Mass Spectrometer Schematic

Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps

High Vacuum System

Inlet

Sample Plate
Target
HPLC
GC
Solids probe

Ion
Source

Mass
Filter

MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI

TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS

Detector

Microch plate
Electron Mult.
Hybrid Detec.

Data
System
PCs
UNIX
Mac

Different Mass Analyzers

Magnetic Sector Analyzer (MSA)

High resolution, exact mass, original MA

Quadrupole Analyzer (Q)

Low (1 amu) resolution, fast, cheap

Time-of-Flight Analyzer (TOF)

No upper m/z limit, high throughput

Ion Trap Mass Analyzer (QSTAR)

Good resolution, all-in-one mass analyzer

Ion Cyclotron Resonance (FT-ICR)

Different Types of MS
ESI-QTOF

Electrospray ionization source + quadrupole

mass filter + time-of-flight mass analyzer

MALDI-QTOF

Matrix-assisted laser desorption ionization +

quadrupole + time-of-flight mass analyzer
Both separate by MW and AA seq

Different Types of MS
GC-MS - Gas Chromatography MS

separates volatile compounds in gas column and IDs

by mass

LC-MS - Liquid Chromatography MS

separates delicate compounds in HPLC column and

IDs by mass

MS-MS - Tandem Mass Spectrometry

separates compound fragments by magnetic field and

IDs by mass

LC/LC-MS/MS-Tandem LC and Tandem MS

Magnetic Sector Analyzer

Quadrupole Mass Analyzer

A quadrupole mass filter consists of four

parallel metal rods with different charges
Two opposite rods have an applied +
potential and the other two rods have a potential
The applied voltages affect the trajectory
of ions traveling down the flight path

For given dc and ac voltages, only ions of

a certain mass-to-charge ratio pass
through the quadrupole filter and all other
ions are thrown out of their original path

Quadrupole Mass Analyzer

Q-TOF Mass Analyzer

NANOSPRAY
TIP

MCP
DETECTOR

PUSHER
HEXAPOLE

QUADRUPOLE
ION
SOURCE

SKIMMER

HEXAPOLE
COLLISION
CELL

TOF
REFLECTRON

HEXAPOLE

Mass Spec Equation (TOF)

2Vt2
m
=
z
L2
m = mass of ion L = drift tube length
z = charge of ion t = time of travel
V = voltage

Ion Trap Mass Analyzer

Ion traps are ion
trapping devices that
make use of a threedimensional quadrupole
field to trap and massanalyze ions
invented by Wolfgang
Paul (Nobel Prize1989)
Offer good mass
resolving power

FT-ICR

Fourier-transform ion cyclotron resonance

Uses powerful magnet (5-10 Tesla) to

create a miniature cyclotron
Originally developed in Canada (UBC) by
A.G. Marshal in 1974
FT approach allows many ion masses to
be determined simultaneously (efficient)
Has higher mass resolution than any other
MS analyzer available

FT-Ion Cyclotron Analzyer

Current Mass Spec Technologies

Proteome profiling/separation
2D SDS PAGE - identify proteins
2-D LC/LC - high throughput analysis of lysates
(LC = Liquid Chromatography)
2-D LC/MS (MS= Mass spectrometry)
Protein identification
Peptide mass fingerprint
Tandem Mass Spectrometry (MS/MS)
Quantative proteomics

ICAT (isotope-coded affinity tag)

ITRAQ

2D - LC/LC
Study protein
complexes
without gel
electrophoresis

Complex mixture is
simplified prior to
MS/MS by 2D LC

(trypsin)

Peptides all bind

to cation
exchange column
Successive elution
with increasing salt
gradients separates
peptides by charge
Peptides are
separated by
hydrophobicity on
reverse phase
column

2D LC/MS

Peptide Mass Fingerprinting

(PMF)

Peptide Mass Fingerprinting

Used to identify protein spots on gels or
protein peaks from an HPLC run
Depends of the fact that if a peptide is cut up
or fragmented in a known way, the resulting
fragments (and resulting masses) are unique
enough to identify the protein
Requires a database of known sequences
Uses software to compare observed masses
with masses calculated from database

Principles of Fingerprinting
Sequence

>Protein 1
acedfhsakdfqea
sdfpkivtmeeewe
ndadnfekqwfe
>Protein 2
acekdfhsadfqea
sdfpkivtmeeewe
nkdadnfeqwfe
>Protein 3
acedfhsadfqeka
sdfpkivtmeeewe
ndakdnfeqwfe

Mass (M+H)

Tryptic Fragments

4842.05

acedfhsak
dfgeasdfpk
ivtmeeewendadnfek
gwfe

4842.05

acek
dfhsadfgeasdfpk
ivtmeeewenk
dadnfeqwfe

4842.05

acedfhsadfgek
asdfpk
ivtmeeewendak
dnfegwfe

Principles of Fingerprinting
Sequence

>Protein 1
acedfhsakdfqea
sdfpkivtmeeewe
ndadnfekqwfe

Mass (M+H)
4842.05

>Protein 2
acekdfhsadfqea
sdfpkivtmeeewe
nkdadnfeqwfe

4842.05

>Protein 3
acedfhsadfqeka
sdfpkivtmeeewe
ndakdnfeqwfe

4842.05

Mass Spectrum

Predicting Peptide Cleavages

http://ca.expasy.org/tools/peptidecutter/

http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

Protease Cleavage Rules

Sometimes
inhibition occurs

Trypsin

XXX[KR]--[!P]XXX

Chymotrypsin
Lys C
Asp N endo
CNBr

XX[FYW]--[!P]XXX

XXXXXK-- XXXXX
XXXXXD-- XXXXX
XXXXXM--XXXXX

K-Lysine, R-Arginine, F-Phenylalanine, Y-Tyrosine,

W-Tryptophan,D-Aspartic Acid, M-Methionine, P-Proline

Why Trypsin?

Robust, stable enzyme

Works over a range of pH values & Temp.
Quite specific and consistent in cleavage
Cuts frequently to produce ideal MW peptides
Inexpensive, easily available/purified
Does produce autolysis peaks (which can be
used in MS calibrations)
1045.56, 1106.03, 1126.03, 1940.94, 2211.10, 2225.12,
2283.18, 2299.18

Digest with specific protease

546 aa

60 kDa; 57 461 Da

pI = 4.75

>RBME00320 Contig0311_1089618_1091255 EC-mopA 60 KDa chaperonin GroEL

MAAKDVKFGR TAREKMLRGV DILADAVKVT LGPKGRNVVI EKSFGAPRIT KDGVSVAKEV
ELEDKFENMG AQMLREVASK TNDTAGDGTT TATVLGQAIV QEGAKAVAAG MNPMDLKRGI
DLAVNEVVAE LLKKAKKINT SEEVAQVGTI SANGEAEIGK MIAEAMQKVG NEGVITVEEA
KTAETELEVV EGMQFDRGYL SPYFVTNPEK MVADLEDAYI LLHEKKLSNL QALLPVLEAV
VQTSKPLLII AEDVEGEALA TLVVNKLRGG LKIAAVKAPG FGDCRKAMLE DIAILTGGQV
ISEDLGIKLE SVTLDMLGRA KKVSISKENT TIVDGAGQKA EIDARVGQIK QQIEETTSDY
DREKLQERLA KLAGGVAVIR VGGATEVEVK EKKDRVDDAL NATRAAVEEG IVAGGGTALL
RASTKITAKG VNADQEAGIN IVRRAIQAPA RQITTNAGEE ASVIVGKILE NTSETFGYNT
ANGEYGDLIS LGIVDPVKVV RTALQNAASV AGLLITTEAM IAELPKKDAA PAGMPGGMGG
MGGMDF

Digest with specific protease

Trypsin yields 47 peptides (theoretically)
Peptide masses in Da:
501.3
674.3
861.4
1000.6
1249.6
1582.9
1790.6
2419.2

533.3
675.4
879.4
1196.6
1249.6
1583.9
1853.9
2526.4

544.3
701.4
921.5
1217.6
1344.7
1616.8
1869.9
2542.4

545.3
726.4
953.4
1228.5
1455.8
1726.7
2286.2
3329.6

614.4
822.4
974.5
1232.6
1484.6
1759.9
2302.2
4211.4

634.3
855.5
988.5
1233.7
1514.8
1775.9
2317.2

http://us.expasy.org/tools/peptide-mass.html

Digest with trypsin

In practice.......see far fewer by mass spec
- possibly incomplete digest (we allow 1 miss)
- lose peptides during each manipulation
washes during digestion
washes during cleanup step
some peptides will not ionize well
some signals (peaks) are poor
low intensity; lack resolution

What Are Missed Cleavages?

Sequence

>Protein 1
acedfhsakdfqea
sdfpkivtmeeewe
ndadnfekqwfe

Tryptic Fragments (no missed cleavage)

acedfhsak (1007.4251)
dfgeasdfpk (1183.5266)
ivtmeeewendadnfek (2098.8909)
gwfe (609.2667)

Tryptic Fragments (1 missed cleavage)

acedfhsak (1007.4251)
dfgeasdfpk (1183.5266)
ivtmeeewendadnfek 2098.8909)
gwfe (609.2667)
acedfhsakdfgeasdfpk (2171.9338)
ivtmeeewendadnfekgwfe (2689.1398)
dfgeasdfpkivtmeeewendadnfek (3263.2997)

Calculating Peptide Masses

Sum the monoisotopic residue masses

Monoisotopic Mass: the sum of the exact or accurate masses of the lightest stable isotope of the atoms
in a molecule

Add mass of H2O (18.01056)

Add mass of H+ (1.00785 to get M+H)
If Met is oxidized add 15.99491
If Cys has acrylamide adduct add 71.0371
If Cys is iodoacetylated add 58.0071
Other modifications are listed at
http://prowl.rockefeller.edu/aainfo/deltamassv2.html

H-1.007828503 amu
2
H-2.014017780 amu
1

C-12
13
C-13.00335, 14C-14.00324
12

Masses in MS
Monoisotopic
mass is the mass
determined using
the masses of the
most abundant
isotopes
Average mass is
the abundance
weighted mass of
all isotopic
components

Mass Calculation (Glycine)

NH2CH2COOH

Amino acid

R1NHCH2COR3

Residue

Monoisotopic Mass
1
H = 1.007825
12
C = 12.00000
14
N = 14.00307
16
O = 15.99491

Glycine Amino Acid Mass

5xH + 2xC + 2xO + 1xN
= 75.032015 amu
Glycine Residue Mass
3xH + 2xC + 1xO + 1xN
=57.021455 amu

Amino Acid Residue Masses

Monoisotopic Mass

Glycine 57.02147
Alanine 71.03712
Serine 87.03203
Proline 97.05277
Valine
99.06842
Threonine 101.04768
Cysteine 103.00919
Isoleucine 113.08407
Leucine 113.08407
Asparagine 114.04293

Aspartic acid 115.02695

Glutamine
128.05858
Lysine
128.09497
Glutamic acid 129.0426
Methionine
131.04049
Histidine
137.05891
Phenylalanine 147.06842
Arginine
156.10112
Tyrosine
163.06333
Tryptophan
186.07932

Amino Acid Residue Masses

Average Mass

Glycine 57.0520
Alanine 71.0788
Serine 87.0782
Proline 97.1167
Valine
99.1326
Threonine 101.1051
Cysteine 103.1448
Isoleucine 113.1595
Leucine 113.1595
Asparagine 114.1039

Aspartic acid 115.0886

Glutamine
128.1308
Lysine
128.1742
Glutamic acid 129.1155
Methionine
131.1986
Histidine
137.1412
Phenylalanine 147.1766
Arginine
156.1876
Tyrosine
163.1760
Tryptophan
186.2133

Preparing a Peptide Mass

Fingerprint Database
Take a protein sequence database (Swiss-Prot or
nr-GenBank)
Determine cleavage sites and identify resulting
peptides for each protein entry
Calculate the mass (M+H) for each peptide
Sort the masses from lowest to highest
Have a pointer for each calculated mass to each
protein accession number in databank

Building A PMF Database

Sequence DB

>P12345
acedfhsakdfqea
sdfpkivtmeeewe
ndadnfekqwfe

Calc. Tryptic Frags

acedfhsak
dfgeasdfpk
ivtmeeewendadnfek
gwfe

>P21234
acekdfhsadfqea
sdfpkivtmeeewe
nkdadnfeqwfe

acek
dfhsadfgeasdfpk
ivtmeeewenk
dadnfeqwfe

>P89212
acedfhsadfqeka
sdfpkivtmeeewe
ndakdnfeqwfe

acedfhsadfgek
asdfpk
ivtmeeewendak
dnfegwfe

Mass List
450.2017 (P21234)
609.2667 (P12345)
664.3300 (P89212)
1007.4251 (P12345)
1114.4416 (P89212)
1183.5266 (P12345)
1300.5116 (P21234)
1407.6462 (P21234)
1526.6211 (P89212)
1593.7101 (P89212)
1740.7501 (P21234)
2098.8909 (P12345)

The Fingerprint (PMF) Algorithm

Take a mass spectrum of a trypsin-cleaved
protein (from gel or HPLC peak)
Identify as many masses as possible in spectrum
(avoid autolysis peaks of trypsin)
Compare query masses with database masses
and calculate # of matches or matching score
(based on length and mass difference)
Rank hits and return top scoring entry this is
the protein of interest

Query (MALDI) Spectrum

1007

1199
2211 (trp)

609
2098

450

1940 (trp)
698

500

1000

1500

2000

2500

Query vs. Database

Query Masses

Database Mass List

450.2201
609.3667
698.3100
1007.5391
1199.4916
2098.9909

450.2017 (P21234)
609.2667 (P12345)
664.3300 (P89212)
1007.4251 (P12345)
1114.4416 (P89212)
1183.5266 (P12345)
1300.5116 (P21234)
1407.6462 (P21234)
1526.6211 (P89212)
1593.7101 (P89212)
1740.7501 (P21234)
2098.8909 (P12345)

Results
2 Unknown masses
1 hit on P21234
3 hits on P12345
Conclude the query
protein is P12345

Database search
PeptIdent (ExPasy)
Mascot (Matrix Science)
MS-Fit (Prospector; UCSF)
ProFound (Proteometrics)
MOWSE (HGMP)
Human Genome Mapping Project

Mascot

800

1200

1600

2000

2400

800

1200

1600

2000

m/z

m/z

theoretical

experimental

Protein ID

2400

What You Need To Do PMF

A list of query masses (as many as possible)
Protease(s) used or cleavage reagents
Databases to search (SWProt, Organism)
Estimated mass and pI of protein spot (opt)

Cysteine (or other) modifications

Minimum number of hits for significance
Mass tolerance (100 ppm = 1000.0 0.1 Da)
A PMF website (Prowl, ProFound, Mascot, etc.)

PMF on the Web

ProFound

http://129.85.19.192/profound_bin/WebProFound.exe

MOWSE

http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse

PeptideSearch

http://www.narrador.emblheidelberg.de/GroupPages/Homepage.html

Mascot

www.matrixscience.com

PeptIdent

http://us.expasy.org/tools/peptident.html

ProFound

ProFound Results

MOWSE

PeptIdent

MASCOT

Mascot Scoring
The statistics of peptide fragment
matching in MS (or PMF) is very similar to
the statistics used in BLAST
The scoring probability follows an extreme
value distribution
High scoring segment pairs (in BLAST)
are analogous to high scoring mass
matches in Mascot
Mascot scoring is much more robust than
arbitrary match cutoffs (like % ID)

Extreme Value Distribution

it is the limit distribution of the maxima of a sequence of independent and

identically distributed random variables. Because of this, the EVD is used as an
approximation to model the maxima of long (finite) sequences of random
variables.

8000
7000

P(x) = 1 - e

6000

-x
-e

5000
4000
3000
2000
1000
0
<20

30

40

50

60

70

80

90

100

110

Scores greater than 72 are significant

>120

MASCOT

Mascot/Mowse Scoring
The Mascot Score is given as S = -10*Log(P),
where P is the probability that the observed
match is a random event
Try to aim for probabilities where P<0.05 (less
than a 5% chance the peptide mass match is
random)
Mascot scores greater than 72 are significant
(p<0.05).

Advantages of PMF

Uses a robust & inexpensive form of MS (MALDI)

Doesnt require too much sample optimization
Can be done by a moderately skilled operator (dont
need to be an MS expert)
Widely supported by web servers
Improves as DBs get larger & instrumentation gets
better
Very amenable to high throughput robotics (up to 500
samples a day)

Limitations With PMF

Requires that the protein of interest
already be in a sequence database
Spurious or missing critical mass peaks
always lead to problems
Mass resolution/accuracy is critical, best
to have <20 ppm mass resolution
Generally found to only be about 40%
effective in positively identifying gel spots

Tandem Mass Spectrometry

Purpose is to fragment ions from parent
ion to provide structural information about
a molecule
Also allows mass separation and AA
identification of compounds in complex
mixtures
Uses two or more mass analyzers/filters
separated by a collision cell filled with
Argon or Xenon
Collision cell is where selected ions are

MS-MS & Proteomics

Tandem Mass Spectrometry

Different MS-MS configurations
Quadrupole-quadrupole (low energy)
Magnetic sector-quadrupole (high)
Quadrupole-time-of-flight (low energy)
Time-of-flight-time-of-flight (low energy)

How Tandem MS
sequencing works
Use Tandem MS: two mass analyzers
in series with a collision cell in
between
Collision cell: a region where the
ions collide with a gas (He, Ne, Ar)
resulting in fragmentation of the ion
Fragmentation of the peptides occur
in a predictable fashion, mainly at
the peptide bonds
The resulting daughter ions have
masses that are consistent with
known molecular weights of
dipeptides, tripeptides,
tetrapeptides

Ser-Glu-Leu-Ile-Arg-Trp

Collision Cell
Ser-Glu-Leu-Ile-Arg
Ser-Glu-Leu-Ile
Ser-Glu-Leu
Etc

Data Analysis Limitations

-You are dependent on well annotated genome
databases
-Data is noisy. The spectra are not always
perfect. Often requires manual determination.
-Database searches only give scores. So if you
have a false positive, you will have to manually
validate them

Advantages of Tandem Mass Spec

FAST
No Gels
Determines MW and AA sequence
Can be used on complex mixtures-including low copy #
Can detect post-translational modif.-ICAT
High-thoughput capability

Disadvantages of Tandem Mass Spec

Very expensive-Campus
Hardware: $1000
Setup: $300
1 run: $1000

Requires sequence databases for analysis

MS-MS & Proteomics

Advantages
Provides precise
sequence-specific data
More informative than
PMF methods (>90%)
Can be used for denovo sequencing (not
entirely dependent on
databases)

Disadvantages
Requires more handling,
refinement and sample
manipulation
Requires more expensive
and complicated
equipment
Requires high level
expertise

Can be used to ID post- Slower, not generally

trans. modifications
high throughput

ISOTOPE-CODED AFFINITY TAG

(ICAT): a quantitative method
Label protein samples with heavy and light reagent
Reagent contains affinity tag and heavy or light isotopes

Chemically reactive group: forms a

covalent bond to the protein or peptide
Isotope-labeled linker: heavy or light,
depending on which isotope is used
Affinity tag: enables the protein or
peptide bearing an ICAT to be isolated by
affinity chromatography in a single step

Example of an ICAT Reagent

Reactive group: Thiol-reactive
group will bind to Cys

Biotin Affinity tag:

Binds tightly to
streptavidin-agarose
resin

Linker: Heavy version will

have deuteriums at *
Light version will have
hydrogens at *

NH

NH

H
N
S

*
*

*
*

H
N

I
O

The ICAT Reagent

How ICAT works?

Affinity isolation
on streptavidin
beads

Lyse &
Label

Quantification
MS
Light

100

100

MIX
Proteolysis
(ie trypsin)

Identification
MS/MS
NH2-EACDPLRCOOH

Heavy

550

570
m/z

590

200

400
m/z

600

ICAT Quantitation

ICAT
Advantages vs. Disadvantages
Estimates relative protein
levels between samples
with a reasonable level of
accuracy (within 10%)

Yield and non specificity

Can be used on complex

mixtures of proteins

Expensive

Cys-specific label reduces

sample complexity
Peptides can be
sequenced directly if
tandem MS-MS is used

Slight chromatography
differences

Tag fragmentation
Meaning of relative
quantification information
No presence of cysteine
residues or not accessible by
ICAT reagent

Mass Spectrometer Schematic

Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps

High Vacuum System

Inlet

Sample Plate
Target
HPLC
GC
Solids probe

Ion
Source

Mass
Filter

MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI

TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS

Detector

Microch plate
Electron Mult.
Hybrid Detec.

Data
System
PCs
UNIX
Mac

MS Detectors

Early detectors used photographic film

Todays detectors (ion channel and electron
multipliers) produce electronic signals via 2o
electronic emission when struck by an ion
Timing mechanisms integrate these signals
with scanning voltages to allow the
instrument to report which m/z has struck the
detector
Need constant and regular calibration

Mass Detectors

Electron Multiplier (Dynode)

Limitations of Proteomics
-solubility of indiv. protein differs
-2D gels unable to resolve all proteins at a given time
-most proteins are not abundant (ie kinases)
-proteins not in the database cannot be identified
-multiple runs can be expensive
-proteins are fragile and can be degraded easily
-proteins exist in multiple isoforms
-no protein equivalent of PCR exists for amplification
of small samples

Shotgun Proteomics:
Multidimensional Protein
Identification Technology
(MudPIT)

General Strategy for Proteomics Characterization

Fractionation &
Isolation
2-DE

Liquid
Chromatography

Peptides

Characterization

Mass Spectrometry

Identification
Post Translational modifications
Quantification

Database Search

MALDI-TOF MS
-(LC)-ESI-MS/MS

Overview of Shotgun Proteomics: MudPIT

Protein Mixture
Digestion

Tandem Mass
Spectrometer

2D Chromatography
RP

MS/MS Spectrum
PySpzS5609 #2438 RT: 66.03 AV: 1 NL: 8.37E6
T: + c d Full ms2 729.75@35.00 [ 190.00-1470.00]
545.31

100
95
90
85
80
75

658.36

70
65

900.36

Relative Abundance

60
55
1031.40
50
45
913.42

40

1240.53
782.23
896.29

35

546.19

771.24

25

1028.41

721.31

20

431.15

15

801.38
559.13
651.14

408.74
399.24

217.91

1241.39

914.34

427.27
317.17

10
5

1032.43

895.33

30

432.40

669.39

1027.22
882.07

600.24

481.13

869.23

915.53
986.50

1258.56

1033.60
1142.43
1123.49

1312.35

1356.10

1195.44

0
200

300

400

500

600

700

800

900
m/z

1000

1100

1200

1300

1400

SEQUEST
DTASelect &
Contrast

SCX

Peptide
Mixture

> 1,000 Proteins

Identified

MudPIT
IEX-HPLC

Trypsin
+ proteins

p53

RP-HPLC

Acquiring MS/MS Datasets

2D Chromatography
SCX

MudPIT Cycle
load sample
wash
salt step
wash
RP gradient
re-equilibration

RP

Tandem MS Spectrum
Peptide Sequence is Inferred from Fragment ions

x 3~18

MS/MS of Peptide Mixtures

LC

MS

(MW Profile)

MS/MS

(AA Identity)

Matching MS/MS Spectra to

Peptide Sequences
SEQUEST

Experimental MS/MS
Spectrum

Peptides Matching Precursor Ion

Mass

Theoretical MS/MS
Spectra

PySpzS5609 #2438 RT: 66.03 AV: 1 NL: 8.37E6

T: + c d Full m s2 729.75@35.00 [ 190.00-1470.00]
545.31

100

#1
CALCULATE #2
#3
#4
#5

95
90
85
80
75
658.36

70
65

900.36

Relative Abundance

60
55
1031.40
50
45
913.42

40

1240.53
782.23
896.29

35

546.19

771.24

25

1028.41

721.31

20

431.15

15

801.38

217.91

559.13
651.14

408.74
399.24

1241.39

914.34

427.27
317.17

10
5

1032.43

895.33

30

432.40

669.39
882.07

600.24

481.13

869.23

K.TVLIMELINNVAK.K
L.NAKMELLIDLVKA.Q
E.ELAILMQNNIIGE.N
A.CGPSRQNLLNAMP.S
L.FAPLQEIINGILE.G

1027.22
915.53
986.50

1258.56

1033.60
1142.43
1123.49

1312.35

1356.10

1195.44

0
200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

m /z

COMPARE
SCORE

SEQUEST Output File

SEQUEST-PVM
Beowolf computing cluster
55 mixed CPU: Alpha chips and AMD
Athlon PC CPU

Filtering, Assembling &

Comparing Protein Lists
20,000s of SEQUEST Output
Files
PARSE

Protein
List

ASSEMBLE

DTASelect

FILTER

Criteria Sets

Contrast
COMPARE
Summary Table
Control

VISUALLY ASSESS SPECTRUM/PEPTIDE MATCHES

Post Analysis Software DTASelect:

Swimming or Drowning in Data

It processes tens of thousands of SEQUEST

outputs in a few minutes.

It applies criteria uniformly and therefore is

unbiased.

It is highly adaptable and re-analysis with a new

set of criteria is easy.

It saves time and effort for manual validation.

The CONTRAST feature can compare results

from different experiments.

Application of shotgun proteomics:

Comprehensive Analysis of Complex
Protein Mixtures

Purification
Cells/Tissues

Multiprotein Complex/
Organelle

Total Protein
Characterization

Yeast: A Perfect Model

Database

ORF

Unknown,
uncoding,

Known,
biochem.

MIPS

6368

hypothetical
1568

or genetics
4344

YPD

6145

1833

4270

SGD

~6000

NA

NA

Complete genome sequence information

An extensively studied organism
Optimal numbers of ORFs, easy for database search

Functional Categories of Yeast Proteins Identified

Used GO to
determine
functional
groups

Communication and Signal Transduction

Ionic Homeostasis

Cell Rescue, Defense, Death, and Ageing

Energy

Cellular Organization

Protein Destination
Transcription
Transport
Protein Synthesis

Metabolism
Unclassified

Cell Growth, Division, DNA synthesis,

and Biogenesis

Washburn et al. Nature Biotechnology 19, 242-7 (2001)

Summary of MudPIT

It is an automated and high throughput

technology.

It is a totally unbias method for protein

identification.

It identifies proteins missed by gel-based

methods (i.e. (low abundance, membrane
proteins etc.)

Post translational modification information of

proteins can be obtained, thus allowing their
functional activities to be derived or inferred.

2-DE vs MudPIT
Widely used, highly
commercialized
High resolving power

Highly automated process

Identified proteins with
extreme pI values, low

abundance and those

from membrane

Visual presentation

Limited dynamic range

Only good for highly soluble
and high abundance proteins
Large amount of sample
required

Thousands of proteins can

be identified

Not yet commercialized

Expensive
Computationally intensive
Quantitation

Peptide Masses From ESI

Each peak is given by:
m/z = (MW + nH+)
n
m/z = mass-to-charge ratio of each peak on spectrum
MW = MW of parent molecule
n = number of charges (integer)
H+ = mass of hydrogen ion (1.008 Da)

Peptide Masses From ESI

Charge (n) is unknown, Key is to determine MW
Choose any two peaks separated by 1 charge
1431.6 = (MW + nH+) 1301.4 = (MW + [n+1]H+)
[n+1]
n
2 equations with 2 unknowns - solve for n first
n = 1300.4/130.2 = 10
Substitute 10 into first equation - solve for MW
MW = 14316 - (10x1.008) = 14305.9

14,305.14

ESI Transformation
Software can be used to convert these
multiplet spectra into single (zero charge)
profiles which gives MW directly
This makes MS interpretation much easier
and it greatly increases signal to noise
Two methods are available

Transformation (requires prior peak ID)

Maximum Entropy (no peak ID required)

Maximum Entropy

ESI and Protein Structure

ESI spectra are actually quite sensitive to
the conformation of the protein
Folded, ligated or complexed proteins tend
to display non-gaussian peak
distributions, with few observable peaks
weighted toward higher m/z values
Denatured or open form proteins/peptides
which ionize easier tend to display many
peaks with a classic gaussian distribution

ESI and Protein Conformation

Native Azurin

Denatured Azurin

Different MS-MS Modes

Product or Daughter Ion Scanning

first analyzer selects ion for further fragmentation

most often used for peptide sequencing

Precursor or Parent Ion Scanning

no first filtering, used for glycosylation studies

Neutral Loss Scanning

selects for ions of one chemical type (COOH, OH)

Selected/Multiple Reaction Monitoring

selects for known, well characterized ions only

THE END