Mass Spectrometry

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Mass Spectrometry Protein Identification

• Micro 343
• David Wishart Rm. Ath 3-41
• david.wishart_at_ualberta.ca

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Objectives

• To learn about the basic principles of mass
  spectrometry, and how a mass spectrometer works
• Calculation of masses for amino acids, residues
  and peptides
• To learn about the principles of peptide mass
  fingerprinting and how this method can be used to
  identify proteins
• Text notes to appear for Friday

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Characterizing Proteins

• Amino Acid Analysis
• Used to identify a protein or peptide based on
  amino acid composition
• Can be used to ID unusual amino acids
• Edman Microsequencing
• Used to determine the N-terminal (5-10 residues)
  of a new protein
• Mass Spectrometry
• State of the art for protein ID

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Mass Spectrometry

• Analytical method to measure the molecular or
  atomic weight of samples

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MS Principles

• Different elements can be uniquely identified by
  their mass

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MS Principles

• Different compounds can be uniquely identified by
  their mass

Butorphanol L-dopa Ethanol
CH3CH2OH
MW 327.1 MW 197.2 MW 46.1
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MS Principles

• Different proteins can be uniquely identified by
  their mass

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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

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Mass Spec Principles
Sample

_
Detector
Ionizer
Mass Analyzer
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Typical Mass Spectrometer
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Typical Mass Spectrum
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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

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Isotopic Distributions
1H 99.9 12C 98.9 35Cl
68.1 2H 0.02 13C 1.1 37Cl
31.9
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Isotopic Distributions
1H 99.9 12C 98.9 35Cl
68.1 2H 0.02 13C 1.1 37Cl
31.9
100
32.1
6.6
2.1
0.06
0.00
m/z
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Mass Calculation (Glycine)
NH2CH2COOH
Amino acid
R1NHCH2COR3
Residue
Glycine Amino Acid Mass 5xH 2xC 2xO 1xN
75.032015 amu Glycine Residue Mass 3xH 2xC
1xO 1xN 57.021455 amu
Monoisotopic Mass 1H 1.007825 12C
12.00000 14N 14.00307 16O 15.99491
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Amino Acid Residue Masses
Monoisotopic Mass
Glycine 57.02147 Alanine 71.03712 Serine 87.03203
Proline 97.05277 Valine 99.06842 Threonine 101.04
768 Cysteine 103.00919 Isoleucine 113.08407 Leucin
e 113.08407 Asparagine 114.04293
Aspartic acid 115.02695 Glutamine 128.05858 Lysin
e 128.09497 Glutamic acid 129.04264 Methionine 1
31.04049 Histidine 137.05891 Phenylalanine 147.06
842 Arginine 156.10112 Tyrosine 163.06333 Trypto
phan 186.07932
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Amino Acid Residue Masses
Average Mass
Glycine 57.0520 Alanine 71.0788 Serine 87.0782 Pro
line 97.1167 Valine 99.1326 Threonine 101.1051 Cy
steine 103.1448 Isoleucine 113.1595 Leucine 113.15
95 Asparagine 114.1039
Aspartic acid 115.0886 Glutamine 128.1308 Lysine
128.1742 Glutamic acid 129.1155 Methionine 131.1
986 Histidine 137.1412 Phenylalanine 147.1766 Arg
inine 156.1876 Tyrosine 163.1760 Tryptophan 186
.2133
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Calculating Peptide Masses

• Sum the monoisotopic residue masses
• Add mass of H2O (18.01056)
• Add mass of H (1.00785 to get MH)
• 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.ht
  ml
• Only consider peptides with masses gt 400

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Inside a Mass Spectrometer
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Mass Spectrometer Schematic
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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

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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

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Soft Ionization Methods
337 nm UV laser
Fluid (no salt)

_
Gold tip needle
cyano-hydroxy cinnamic acid
MALDI
ESI
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MALDI Ionization
Matrix

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

-
-
Laser
-

Analyte



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-

-

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MALDI Spectra (Mass Fingerprint)
Tumor
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Mass Spectrometer Schematic
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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)
• Highest resolution, exact mass, costly

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Quadrupole Mass Analyzer

• A quadrupole mass filter consists of four
  parallel metal rods with different charges
• Two opposite rods have an applied potential of
  (UVcos(wt)) and the other two rods have a
  potential of -(UVcos(wt))
• 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

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Q-TOF Mass Analyzer
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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
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Peptide Mass Fingerprinting (PMF)
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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

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Principles of Fingerprinting
Sequence Mass (MH) Tryptic Fragments
gtProtein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekq
wfe gtProtein 2 acekdfhsadfqea sdfpkivtmeeewe nkda
dnfeqwfe gtProtein 3 acedfhsadfqeka sdfpkivtmeeewe
ndakdnfeqwfe
acedfhsak dfgeasdfpk ivtmeeewendadnfek gwfe
acek dfhsadfgeasdfpk ivtmeeewenk dadnfeqwfe ace
dfhsadfgek asdfpk ivtmeeewendak dnfegwfe
4842.05 4842.05 4842.05
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Principles of Fingerprinting
Sequence Mass (MH) Mass Spectrum
gtProtein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekq
wfe gtProtein 2 acekdfhsadfqea sdfpkivtmeeewe nkda
dnfeqwfe gtProtein 3 acedfhsadfqeka sdfpkivtmeeewe
ndakdnfeqwfe
4842.05 4842.05 4842.05
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Predicting Peptide Cleavages
http//ca.expasy.org/tools/peptidecutter/
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http//ca.expasy.org/tools/peptidecutter/peptidecu
tter_enzymes.htmlTryps
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Protease Cleavage Rules
Trypsin XXXKR--!PXXX Chymotrypsin XXFYW--
!PXXX Lys C XXXXXK-- XXXXX Asp N
endo XXXXXD-- XXXXX CNBr XXXXXM--XXXXX
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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

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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 (MH) for each peptide
• Sort the masses from lowest to highest
• Have a pointer for each calculated mass to each
  protein accession number in databank

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Building A PMF Database
Sequence DB Calc. Tryptic Frags Mass List
gtP12345 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe
gtP21234 acekdfhsadfqea sdfpkivtmeeewe nkdadnfeqw
fe gtP89212 acedfhsadfqeka sdfpkivtmeeewe ndakdnfe
qwfe
acedfhsak dfgeasdfpk ivtmeeewendadnfek gwfe
acek dfhsadfgeasdfpk ivtmeeewenk dadnfeqwfe ace
dfhsadfgek asdfpk ivtmeeewendak dnfegwfe
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)
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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)
• 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

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Query (MALDI) Spectrum
1007
1199
2211 (trp)
609
2098
450
1940 (trp)
698
500 1000 1500 2000
2500
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Query vs. Database
Query Masses Database Mass List
Results
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)
450.2201 609.3667 698.3100 1007.5391 1199.4916 209
8.9909
2 Unknown masses 1 hit on P21234 3 hits on
P12345 Conclude the query protein is P12345
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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.)

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PMF on the Web

• Mascot
• www.matrixscience.com
• ProFound
• http//129.85.19.192/profound_bin/WebProFound.exe
• MOWSE
• http//srs.hgmp.mrc.ac.uk/cgi-bin/mowse
• PeptideSearch
• http//www.narrador.embl-heidelberg.de/GroupPages/
  Homepage.html
• PeptIdent
• http//us.expasy.org/tools/peptident.html

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ProFound
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ProFound (PMF)
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What Are Missed Cleavages?
Sequence Tryptic Fragments (no missed cleavage)
gtProtein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekq
wfe
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) ivtmeee
wendadnfekgwfe (2689.1398) dfgeasdfpkivtmeeewendad
nfek (3263.2997)
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ProFound Results
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Mascot PMF Query
click
http//www.matrixscience.com/search_form_select.ht
ml
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MASCOT
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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)

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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 lt20 ppm mass resolution
• Generally found to only be about 40 effective in
  positively identifying gel spots or HPLC peaks