Mass Spectrometry: Methods

1
Mass Spectrometry Methods Theory
2
Proteomics Tools

• Molecular Biology Tools
• Separation Display Tools
• Protein Identification Tools
• Protein Structure Tools

3
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

4
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

5
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

6
How does a mass spectrometer work?
Create ions
Separate ions
Detect ions

• Mass spectrum
• Database analysis

• Ionization method
• MALDI
• Electrospray
• (Proteins must be charged and dry)

• Mass analyzer
• MALDI-TOF
• MW
• Triple Quadrapole
• AA seq
• MALDI-QqTOF
• AA seq and MW
• QqTOF
• AA seq and protein modif.

7
Generalized Protein Identification by MS
Spectrum of fragments generated
MATCH
Library
Database of sequences (i.e. SwissProt)
8
Methods for protein identification
9
MS Principles

• Different elements can be uniquely identified by
  their mass

10
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
11
Mass Spectrometry

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

12
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 e-
• It then analyzes those ions to provide
  information about the molecular
• weight of the compound and its chemical
  structure.

13
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)

14
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

15
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

16
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

17
Mass Spec Principles
Sample

_
Detector
Ionizer
Mass Analyzer
18
How does a mass spectrometer work?
Create ions
Separate ions
Detect ions

• Mass spectrum
• Database analysis

• Ionization method
• MALDI
• Electrospray
• (Proteins must be charged and dry)

• Mass analyzer
• MALDI-TOF
• MW
• Triple Quadrapole
• AA seq
• MALDI-QqTOF
• AA seq and MW
• QqTOF
• AA seq and protein modif.

19
Mass spectrometers

• 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
  from multiple laser bursts are averaged. Multiple
  laser
• 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
• 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)

20
Typical Mass Spectrometer
21
LC/LC-MS/MS-Tandem LC, Tandem MS
22
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)

23
m/z ratio
All proteins are sorted based on a mass to
charge ratio (m/z)

• Molecular weight divided by the charge on this
  protein

24
Typical Mass Spectrum
Relative Abundance
aspirin
120 m/z-for singly charged ion this is the mass
25
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 is the mass number of the observed mass (DM) is
  the difference between two masses that can be
  separated

26
Resolution in MS
27
Resolution in MS
783.455
QTOF
784.465
785.475
783.6
28
Mass Spectrometer Schematic
29
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

30
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 gtgt
  5 eV bonds)
• Fragments sent to mass analyzer

31
(No Transcript)
32
EI Fragmentation of CH3OH
CH3OH
CH3OH
CH3OH
CH2OH
H
CH3OH
CH3
OH
H
CH2OH
CHOH
Why wouldnt Electron Impact be suitable for
analyzing proteins?
33
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 sub-fragments and
  even peptides of 2,3,4 amino acids
• Result is 10,000s of different signals from a
  single protein -- too complex to analyze

34
Soft Ionization Methods
337 nm UV laser
Fluid (no salt)

_
Gold tip needle
cyano-hydroxy cinnamic acid
MALDI
ESI
35
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

36
(No Transcript)
37
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.
• Matrix-assisted laser desorption ionization
  (MALDI)
• Analyte (protein) is mixed with large excess of
  matrix (small organic molecule)
• Irradiated with short pulse of laser light.
  Wavelength of laser is the same as absorbance max
  of matrix.

38
Electrospray Ionization

• Sample dissolved in polar, volatile buffer (no
  salts) and pumped through a stainless steel
  capillary (70 - 150 mm) at a rate of 10-100
  mL/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)

39
Electrospray (Detail)
40
Electrospray Ionization

• Can be modified to nanospray system with flow lt
  1 mL/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)

41
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

42
Matrix-Assisted Laser Desorption Ionization
337 nm UV laser
cyano-hydroxy cinnamic acid
MALDI
43
MALDI

• Sample is ionized by bombarding sample with laser
  light
• Sample is mixed with a UV absorbant matrix
  (sinapinic acid for proteins, 4-hydroxycinnaminic
  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)

44
HT Spotting on a MALDI Plate
45
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



-

-

-

-






46
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
• Requires 10 mL of 1 pmol/mL sample

47
Principal for MALDI-TOF MASS
48
Principal for MALDI-TOF MASS
49
MALDI SELDI
337 nm UV laser
cyano-hydroxy cinnaminic acid
MALDI
50
MALDI/SELDI Spectra
Normal
Tumor
51
Mass Spectrometer Schematic
52
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

53
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

54
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
• Separates by HPLC, IDs by mass and AA sequence

55
Magnetic Sector Analyzer
56
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

57
Quadrupole Mass Analyzer
58
Q-TOF Mass Analyzer
59
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
60
Ion Trap Mass Analyzer

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

61
FT-ICRFourier-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

62
FT-Ion Cyclotron Analzyer
63
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

64
2D - LC/LC
Peptides all bind to cation exchange column
(trypsin)
Study protein complexes without gel
electrophoresis
Successive elution with increasing salt gradients
separates peptides by charge
Peptides are separated by hydrophobicity on
reverse phase column
Complex mixture is simplified prior to MS/MS by
2D LC
65
2D - LC/MS
66
Peptide Mass Fingerprinting (PMF)
67
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

68
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
69
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
70
Predicting Peptide Cleavages
http//ca.expasy.org/tools/peptidecutter/
71
http//ca.expasy.org/tools/peptidecutter/peptidecu
tter_enzymes.htmlTryps
72
Protease Cleavage Rules
Sometimes inhibition occurs
Trypsin XXXKR--!PXXX Chymotrypsin XXFYW--
!PXXX Lys C XXXXXK-- XXXXX Asp N
endo XXXXXD-- XXXXX CNBr XXXXXM--XXXXX
K-Lysine, R-Arginine, F-Phenylalanine,
Y-Tyrosine, W-Tryptophan,D-Aspartic Acid,
M-Methionine, P-Proline
73
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

74
Digest with specific protease
546 aa 60 kDa 57 461 Da pI 4.75
gtRBME00320 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
75
Digest with specific protease
Trypsin yields 47 peptides (theoretically)
Peptide masses in Da
501.3 533.3 544.3 545.3 614.4 634.3 674.3 675.4
701.4 726.4 822.4 855.5 861.4 879.4 921.5 953.4
974.5 988.5 1000.6 1196.6 1217.6 1228.5 1232.6 1
233.7 1249.6 1249.6 1344.7 1455.8 1484.6 1514.8
1582.9 1583.9 1616.8 1726.7 1759.9 1775.9 1790
.6 1853.9 1869.9 2286.2 2302.2 2317.2 2419.2 252
6.4 2542.4 3329.6 4211.4
http//us.expasy.org/tools/peptide-mass.html
76
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
77
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)
78
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 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
• 1H-1.007828503 amu 12C-12
• 2H-2.014017780 amu 13C-13.00335, 14C-14.00324

79
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

80
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
81
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 129.0426 Methionine 13
1.04049 Histidine 137.05891 Phenylalanine 147.068
42 Arginine 156.10112 Tyrosine 163.06333 Tryptop
han 186.07932
82
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
83
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

84
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)
85
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

86
Query (MALDI) Spectrum
1007
1199
2211 (trp)
609
2098
450
1940 (trp)
698
500 1000 1500 2000
2500
87
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)
2 Unknown masses 1 hit on P21234 3 hits on
P12345 Conclude the query protein is P12345
450.2201 609.3667 698.3100 1007.5391 1199.4916 209
8.9909
88
Database search
Mascot
theoretical
experimental
Protein ID
89
(No Transcript)
90
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.)

91
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.embl-heidelberg.de/GroupPages/
  Homepage.html
• Mascot
• www.matrixscience.com
• PeptIdent
• http//us.expasy.org/tools/peptident.html

92
ProFound
93
ProFound Results
94
MOWSE
95
PeptIdent
96
MASCOT
97
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)

98
Extreme Value Distributionit 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.
Scores greater than 72 are significant
99
MASCOT
100
Mascot/Mowse Scoring

• The Mascot Score is given as S -10Log(P),
  where P is the probability that the observed
  match is a random event
• Try to aim for probabilities where Plt0.05 (less
  than a 5 chance the peptide mass match is
  random)
• Mascot scores greater than 72 are significant
  (plt0.05).

101
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)

102
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

103
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 sent
  for further fragmentation

104
MS-MS Proteomics
105
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)

106
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
107
(No Transcript)
108
(No Transcript)
109
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
110
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-thoughp
ut capability
Disadvantages of Tandem Mass Spec
Very expensive-Campus Hardware 1000 Setup
300 1 run 1000 Requires sequence databases for
analysis
111
MS-MS Proteomics
Advantages Disadvantages

• Provides precise sequence-specific data
• More informative than PMF methods (gt90)
• Can be used for de-novo sequencing (not entirely
  dependent on databases)
• Can be used to ID post-trans. modifications

• Requires more handling, refinement and sample
  manipulation
• Requires more expensive and complicated equipment
• Requires high level expertise
• Slower, not generally high throughput

112
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
113
Example of an ICAT Reagent
Biotin Affinity tag Binds tightly to
streptavidin-agarose resin
Reactive group Thiol-reactive group will bind to
Cys
Linker Heavy version will have deuteriums at
Light version will have hydrogens at
114
The ICAT Reagent
115
How ICAT works?
Affinity isolation on streptavidin beads
Lyse Label
Quantification MS
Identification MS/MS
NH2-EACDPLR-COOH
Light
100
MIX
Heavy
Proteolysis (ie trypsin)
m/z
m/z
116
ICAT Quantitation
117
ICATAdvantages vs. Disadvantages

• Estimates relative protein levels between samples
  with a reasonable level of accuracy (within 10)
• Can be used on complex mixtures of proteins
• Cys-specific label reduces sample complexity
• Peptides can be sequenced directly if tandem
  MS-MS is used

• Yield and non specificity
• Slight chromatography differences
• Expensive
• Tag fragmentation
• Meaning of relative quantification information
• No presence of cysteine residues or not
  accessible by ICAT reagent

118
Mass Spectrometer Schematic
119
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

120
Mass Detectors
Electron Multiplier (Dynode)
121
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
122
(No Transcript)
123
General Strategy for Proteomics Characterization
Fractionation Isolation
Liquid Chromatography
2-DE
Peptides
Mass Spectrometry
Database Search
124
Overview of Shotgun Proteomics MudPIT
Protein Mixture
Digestion
Tandem Mass Spectrometer
Peptide Mixture
gt 1,000 Proteins Identified
SEQUEST DTASelect Contrast
MS/MS Spectrum
125
MudPIT
IEX-HPLC
RP-HPLC
Trypsin proteins
p53
126
Acquiring MS/MS Datasets
Tandem MS Spectrum Peptide Sequence is Inferred
from Fragment ions
127
MS/MS of Peptide Mixtures
LC
MS (MW Profile)
MS/MS (AA Identity)
128
Matching MS/MS Spectra to Peptide Sequences
SEQUEST
Experimental MS/MS Spectrum
Peptides Matching Precursor Ion Mass
Theoretical MS/MS Spectra
1 K.TVLIMELINNVAK.K 2 L.NAKMELLIDLVKA.Q 3
E.ELAILMQNNIIGE.N 4 A.CGPSRQNLLNAMP.S 5
L.FAPLQEIINGILE.G
CALCULATE
COMPARE
SCORE
SEQUEST Output File
129
SEQUEST-PVM
Beowolf computing cluster 55 mixed CPU Alpha
chips and AMD Athlon PC CPU
130
Filtering, Assembling Comparing Protein Lists
20,000s of SEQUEST Output Files
Protein List
ASSEMBLE
PARSE
DTASelect
FILTER
Criteria Sets
Contrast
COMPARE
Summary Table
VISUALLY ASSESS SPECTRUM/PEPTIDE MATCHES
131
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.

132
(No Transcript)
133
Application of shotgun proteomics Comprehensive
Analysis of ComplexProtein Mixtures
Total Protein Characterization
134
Yeast A Perfect Model

• Complete genome sequence information
• An extensively studied organism
• Optimal numbers of ORFs, easy for database search

135
Functional Categories of Yeast Proteins Identified
Used GO to determine functional groups
Washburn et al. Nature Biotechnology 19, 242-7
(2001)
136
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.

137
2-DE vs MudPIT

• Widely used, highly commercialized
• High resolving power
• Visual presentation
• Limited dynamic range
• Only good for highly soluble and high abundance
  proteins
• Large amount of sample required

• Highly automated process
• Identified proteins with extreme pI values, low
  abundance and those from membrane
• Thousands of proteins can be identified
• Not yet commercialized
• Expensive
• Computationally intensive
• Quantitation

138
Peptide Masses From ESI
Each peak is given by
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)
139
Peptide Masses From ESI
Charge (n) is unknown, Key is to determine
MW Choose any two peaks separated by 1 charge
1301.4 (MW n1H)
1431.6 (MW nH)
n1
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
140
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)

141
Maximum Entropy
142
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

143
ESI and Protein Conformation
Native Azurin
Denatured Azurin
144
(No Transcript)
145
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

146
THE END