Mass Spectrometry-Based Methods for Protein Identification

1
Mass Spectrometry-Based Methods for Protein
Identification
Joseph A. Loo Department of Biological
Chemistry David Geffen School of
Medicine Department of Chemistry and
Biochemistry University of California Los
Angeles, CA USA
2
Genomics and ProteomicsCharacterizing many genes
and gene products simultaneously
3
Proteomics Aids Biological Research
complex protein mixture
Biology
protein identification protein modification protei
n abundance
protein separation
mass spectrometry
4
Proteomics - What is it?

• An assay to systematically analyze the diverse
  properties of proteins
• Biological processes are dynamic
• A quantitative comparison of states is required
• The study of protein expression and function on a
  genome scale
• Purpose Examine altered gene expression
  pathways in disease states and under different
  environmental conditions

The completion of the human genome has provided
researchers with the blueprint for life, and
proteomics offers scientists the means for
analyzing the expressed genome.
5
Genome to Proteome
dsDNA
(Gene)
Transcription
mRNA
Translation
Protein
H2N
COOH
MTDLKASSLRALKLMDLTTLNDDDTDEKVIALCHQAKTPVGNTA
AICIYP 51 RFIPIARKTLKEQGTPEIRIATVTNFPHGNDDIDIAL
AETRAAIAYGADE 101 VDVVFPYRALMAGNEQVGFDLVKACKEACA
AANVLLKVIIETGELKDEAL 151 IRKASEISI
Mass spectrometry
The completion of the human genome has provided
researchers with the blueprint for life, and
proteomics offers scientists the means for
analyzing the expressed genome.
6
Approaches for Protein Identification
What is this protein?

• Molecular weight
• Isoelectric point
• Amino acid composition
• Other physical/chemical characteristics
• Partial or complete amino acid sequence
• Edman (N-terminal sequence) - if N-term. not
  blocked
• C-terminal sequence - not commonly performed
• Mass spectrometry-measured information

7
Protein Identification by Mass Spectrometry
2-D Gel Electrophoresis
150-
75-
40-
25-
MW x 103
18-
10-
6.5
6.0
5.5
5.0
4.5
pI
1547
1089
Peptide mass fingerprint by MALDI-TOF or
LC-ESI-MS. Additional sequence information can
be obtained by MS/MS.
2384
717
1857
1401
1700
1272
2791
500
2500
3000
1500
m/z
8
Mass SpectrometryA method to weigh molecules
A simple measurement of mass is used to confirm
the identity of a molecule, but it can be used
for much more
9
Mass Spectrometer for Proteomics
Pre-Separation
Ion Source
Liquid Chromatography
10
The Nobel Prize in Chemistry 2002
"for the development of methods for
identification and structure analyses of
biological macromolecules"
"for their development of soft desorption
ionisation methods for mass spectrometric
analyses of biological macromolecules"
11
Electrospray Generation of aerosols and droplets
12
Electrospray Ionization (ESI)

• Multiple charging
• More charges for larger molecules
• MW range gt 150 kDa
• Liquid introduction of analyte
• Interface with liquid separation methods, e.g.
  liquid chromatography
• Tandem mass spectrometry (MS/MS) for protein
  sequencing

ESI
MS
high voltage
highly charge droplets
20
19
18
21
17
16
22
15
14
500
700
900
1100
mass/charge (m/z)
13
ESI-MS of Large Proteins
distribution of multiply charged molecules
(M14H)14
(M15H)15
3323
3102
(M13H)13
3543
(M16H)16
ESI-MS (Q-TOF) pH 7.5
m/z
14
History of Electrospray Ionization

• Malcolm Dole demonstrated the production of
  intact oligomers of polystyrene up to MW 500,000
• mass analysis of large ions was problematic
• John Fenn (Yale University)
• Chemical engineer - expert in supersonic
  molecular beams
• Began work on electrospray in 1981
• Adapted ESI to operate on a more conventional
  mass spectrometer
• Recognized that multiply charged ions were
  produced by ESI
• Reduced the m/z range required

15
Electrospray process
106 charges for 30 micron droplet

• Analyte dissolved in a suitable solvent flows
  through a small diameter capillary tube
• Liquid in the presence of a high electric field
  generates a fine mist or aerosol spray of
  highly charged droplets

16
Matrix-assisted Laser Desorption/Ionization
(MALDI)
Time-of-Flight (TOF) Analyzer
detector
high voltage
MALDI
sample
laser
drift region
m1 m2 m3
17
MALDI Mass Spectrometry of Large Proteins
100
97430
MALDI-MS of rat MVP
(MH)
Intensity
(M2H)2
98563
48658
36446
58309
30811
50608
70405
90202
110000
129797
m/z
18
MALDI
sample and matrix

• Developed by Tanaka (Japan) and Hillenkamp/Karas
  (Germany)
• Peptide/protein analyte of interest is
  co-crystallized on the MALDI target plate with an
  appropriate matrix
• small, highly conjugated organic molecules which
  strongly absorb energy at a particular wavelength
• Energy is transferred to analyte indirectly,
  inducing desorption from target surface
• Analyte is ionized by gas-phase proton transfer
  (perhaps from ionized matrix molecules)

pulsed laser light
peptide/protein ions desorbed from matrix
20 kV (sample stage or target)
19
MALDI matrices
2,5-dihydroxybenzoic acid (DHB) peptides and
proteins
4-hydroxy-?-cyanocinnamic acid (alpha-cyano or
4-HCCA) peptides
3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic
acid) proteins
matrices for 337 nm irradiation
20
MALDI

• 337 nm irradiation is provided by a nitrogen (N2)
  laser
• The target plate is inserted into the high vacuum
  region of the source and the sample is irradiated
  with a laser pulse. The matrix absorbs the laser
  energy and transfers energy to the analyte
  molecule. The molecules are desorbed and ionized
  during this stage of the process.
• MALDI is most commonly interfaced to a
  time-of-flight (TOF) mass spectrometer.

21
R. Aebersold and M. Mann, Nature (2003), 422,
198-207.
22
Time-of-Flight Mass Spectrometer
v1 m1
v2 m2
v3 m3
detector
drift region (L)
high voltage
Principal of Operation of Linear TOF A
time-of-flight mass spectrometer measures the
mass-dependent time it takes ions of different
masses to move from the ion source to the
detector. This requires that the starting time
(the time at which the ions leave the ion source)
is well-defined. Recall that the kinetic energy
of an ion is
where ? is ion velocity, m is mass, e is
charge on electron, and V is electric field.
The ion velocity, ?, is also the length of the
flight path, L , divided by the flight time, t
Substituting this expression for ? into the
kinetic energy relation, we can derive the
working equation for the time-of-flight mass
spectrometer
mass is proportional to (time)2
23
Approaches for Protein Sequencing and
Identification
Top Down
MS/MS
MIRERICACVLALGMLTGFTHAFGSKDAAADGKPLVVTTIGMIADAVKNI
AQGDVHLKGLMGPGVDPHLYTATAGDVEWLGNADLILYNGLHLETKMGEV
FSKLRGSRLVVAVSETIPVSQRLSLEEAEFDPHVWFDVKLWSYSVKAVYE
SLCKLLPGKTREFTQRYQAYQQQLDKLDAYVRRKAQSLPAERRVLVTAHD
AFGYFSRAYGFEVKGLQGVSTASEASAHDMQELAAFIAQRKLPAIFIESS
IPHKNVEALRDAVQARGHVVQIGGELFSDAMGDAGTSEGTYVGMVTHNID
TIVAALAR
MS/MS
Enzymatic or chemical degradation
Bottom Up
24
Identification of proteins from gels

• Proteins are separated first by high resolution
  two-dimensional polyacrylamide gel
  electrophoresis and then stained. At this point,
  to identify an individual or set of protein
  spots, several options can be considered by the
  researcher, depending on availability of
  techniques.
• For protein spots that appear to be relatively
  abundant (e.g., more than 1 pmol), traditional
  protein characterization methods may be employed.
• Methods such as amino acid analysis and Edman
  sequencing can be used to provide necessary
  protein identification information. With 2-DE,
  approximate molecular weight and isoelectric
  point characteristics are provided. Augmented
  with information on amino acid composition and/or
  amino-terminal sequence, a confident
  identification can be obtained.
• The sensitivity gains of using MS allows for the
  identification of proteins below the one pmol
  level and in many cases in the femtomole regime.

25
Protein Identification by Mass Spectrometry
2-D Gel Electrophoresis
150-
75-
40-
25-
MW x 103
18-
10-
6.5
6.0
5.5
5.0
4.5
pI
1547
1089
Peptide mass fingerprint by MALDI-TOF or
LC-ESI-MS. Additional sequence information can
be obtained by MS/MS.
2384
717
1857
1401
1700
1272
2791
500
2500
3000
1500
m/z
26
Protein Cleavage

• For the application of mass spectrometry for
  protein identification, the protein bands/spots
  from a 2-D gel are excised and are exposed to a
  highly specific enzymatic cleavage reagent (e.g.,
  trypsin cleaves on the C-terminal side of
  arginine and lysine residues). The resulting
  tryptic fragments are extracted from the gel
  slice and are then subjected to MS-methods. One
  of the major barriers to high throughput in the
  proteomic approach to protein identification is
  the in-gel proteolytic digestion and subsequent
  extraction of the proteolytic peptides from the
  gel. Common protocols for this process are often
  long and labor intensive.

protein digestion robot
27
Protein cleavage - proteolysis and chemical
methods
28
Mass spectrometry-based protein identification

• A mass spectrum of the resulting digest products
  produces a peptide map or a peptide
  fingerprint.
• The measured masses can be compared to
  theoretical peptide maps derived from database
  sequences for identification. There are a few
  choices of mass analysis that can be selected
  from this point, depending on available
  instrumentation and other factors. The resulting
  peptide fragments can be subjected to MALDI-MS or
  ESI-MS analysis.
• A small aliquot of the digest solution can be
  directly analyzed by MALDI-MS to obtain a peptide
  map. The resulting sequence coverage (relative
  to the entire protein sequence) displayed from
  the total number of tryptic peptides observed in
  the MALDI mass spectrum can be quite high, i.e.,
  greater than 80 of the sequence, although it can
  vary considerably depending on the protein,
  sample amount, etc. The measured molecular
  weights of the peptide fragments along with the
  specificity of the enzyme employed can be
  searched and compared against protein sequence
  databases using a number of computer searching
  routines available on the Internet.

29
Protein identification from peptide fragments
Tryptic peptides
Mass spectrum
Protein
Theoretical mass spectrum
Theoretical tryptic peptides
Protein sequence
SEMHIKHYTTK ILGFR EEGDSCPLK QWDDSK ILVAVADK LLEYEE
K ILLFNSAK YLLDESSTYK LMHDDSV
SEMHIKHYTTKILGFREEGDSCPLKQWDDSKILVAVADKLLEYEEKILLF
NSAKYLLDESSTYKLMHDDSV
30
MALDI-MS of tryptic peptides
1247.70
all peaks are (MH)
1116.67
1375.76
trypsin autolysis
1505.77
1424.85
1665.89
2005.07
1287.73

2719.48
1574.20
1811.85

1849.12
2476.21
2550.52
1000
1500
2000
2500
3000
m/z
ARIIVVTSGK GGVGKTTSSA AIATGLAQKG KKTVVIDFDI
GLRNLDLIMG CERRVVYDFV NVIQGDATLN QALIKDKRTE
NLYILPASQT RDKDALTREG VAKVLDDLKA MDFEFIVCDS
PAGIETGALM ALYFADEAII TTNPEVSSVR DSDRILGILA
SKSRRAENGE EPIKEHLLLT RYNPGRVSRG DMLSMEDVLE
ILRIKLVGVI PEDQSVLRAS NQGEPVILDI NADAGKAYAD
TVERLLGER PFRFIEEEKK GFLKRLFGG
31
ESI-MS and LC-MS for protein identification

• An approach for peptide mapping similar to
  MALDI-MS uses ESI-MS. A peptide map can be
  obtained by analysis of the peptide mixture by
  ESI-MS. An advantage of ESI is its ease of
  coupling to separation methodologies such as
  HPLC. Thus, alternatively, to reduce the
  complexity of the mixture, the peptides can be
  separated by HPLC with subsequent mass
  measurement by on-line ESI-MS. The measured
  masses can be compared to sequence databases.

9.4
LC-MS with ESI
8.4
9.8
8.9
7.7
6.8
6.2
Time (min)
965.3
(M2H)2
MW 1928.6 Da
629.0
m/z
32
LC-MS/MS for protein identification

• An improvement in throughput of the overall
  method can be obtained by performing LC-MS/MS in
  the data dependant mode. As full scan mass
  spectra are acquired continuously in LC-MS mode,
  any ion detected with a signal intensity above a
  pre-defined threshold will trigger the mass
  spectrometer to switch over to MS/MS mode. Thus,
  the mass spectrometer switches back and forth
  between MS- (molecular mass information) and
  MS/MS mode (sequence information) in a single LC
  run. The data dependant scanning capability can
  dramatically increase the capacity and throughput
  for protein identification.

9.4
y12
8.4
LC-MS
LC-MS/MS
1261.4
9.8
y10
6.8
y13
1374.5
Time (min)
b6
668.4
b8
965.3
838.5
b5
y9
y11
y14
(M2H)2
MS/MS
y8
1474.4
y4
629.0
b3
m/z
m/z
33
Peptide sequencing by mass spectrometry
N-term.
C-term.

• Peptide molecules are fragmented by collisionally
  activated dissociation (CAD)
• collisions with neutral background gas molecules
  (nitrogen, argon, etc)
• typically dissociate by cleavage of -CO-NH- bond

A
N-terminal product ions
34
Peptide sequencing by mass spectrometry

• Ideally, one can measure the spacings between
  product ion peaks to deduce the sequence
• if each amide bond dissociates with equal
  probability
• if only a single amide bond fragments for each
  molecule
• if only C-terminal or N-terminal products ions
  are formed
• In reality, this is not the case

C-terminal product ions
35
Nomenclature for MS Sequencing of Peptides
Klaus Biemann, MIT
subscript denotes the number of residues
contained in product ion
N-terminal fragments
b1
b2
b3
H2N - C - C - N - C - C - N - C - C - N - C - COOH
y3
y2
y1
C-terminal fragments
36
Nomenclature for MS Sequencing of Peptides

• Low-energy collisions promote fragmentation of a
  peptide primarily along the peptide backbone
• Peptide fragmentation which maintains the charge
  on the C terminus is designated a y-ion
• Fragmentation which maintains the charge on the N
  terminus is designated a b-ion
• Low energy collisions ion trap, QQQ, QTOF,
  FT-ICR
• High energy collisions TOF-TOF
• cleavage of amino acid side chain bonds (d-ion
  and w-ion)
• differentiate Leu vs. Ile

37
Peptide Sequencing by Mass Spectrometry
y4-14
LVDKVIGITNEEAISTAR
Cysteine Synthase A
b3-17
MS/MS of 2 charged tryptic peptides yield
(often) 1 charged product ions (but 2 charged
products can be observed as well)
y12
1261.4
y10
mixture of b-ions and y-ions are present
1091.5
y13
1374.5
Rel. Abund.
b6
668.4
b8
b5
y9
838.5
y14
555.4
990.5
y11
y5
b7
1474.4
y4
b4
b9
y6
y8
b3
y7
b14
b10
b11
b12
b13
b16
b17
b15
m/z
38
Computer-based Sequence Searching Strategies

• A list of experimentally determined masses is
  compared to lists of computer-generated
  theoretical masses prepared from a database of
  protein primary sequences. With the current
  exponential growth in the generation of genomic
  data, these databases are expanding every day.
• There are typically three types of search
  strategies employed
• searching with peptide fingerprint data
• searching with sequence data
• searching with raw MS/MS data.
• One limiting factor that must be considered for
  all of the approaches is that they can only
  identify proteins that have been identified and
  reside within an available database, or very
  homologous to one that resides in the database.

39
Searching with Peptide Fingerprints

• The majority of the available search engines
  allow one to define certain experimental
  parameters to optimize a particular search.
• Minimum number of peptides to be matched
• Allowable mass error
• Monoisotopic versus average mass data
• Mass range of starting protein
• Type of protease used for digestion
• Information about potential protein modification,
  such as N- and C-terminal modification,
  carboxymethylation, oxidized methionines, etc.

40
Searching with Peptide Fingerprints

• Most protein databases contain primary sequence
  information only
• Any shift in mass incorporated into the primary
  sequence as a result of post-translational
  modification will result in an experimental mass
  that is in disagreement with the theoretical
  mass.
• Modifications such as glycation and
  phosphorylation can result in missed
  identifications.
• A single amino acid substitution can shift the
  mass of a peptide to such a degree that even a
  protein with a great deal of homology with
  another in the database can not be identified.

41
Searching with Peptide Fingerprints

• A number of factors affect the utility of peptide
  fingerprinting.
• The greater the experimental mass accuracy, the
  narrower you can set your search tolerances,
  thereby increasing your confidence in the match,
  and decreasing the number of "false positive"
  responses.
• A common practice used to increase mass accuracy
  in peptide fingerprinting is to employ an
  autolysis fragment from the proteolytic enzyme as
  an internal standard to calibrate a MALDI mass
  spectrum.
• Peptide fingerprinting is also amenable to the
  identification of proteins in complex mixtures.
• Peptides generated from the digest of a protein
  mixture will simply return two or more results
  that are a "good" fit.
• Peptides that are "left over" in a peptide
  fingerprint after the identification of one
  component can be resubmitted for the possible
  identification of another component.

42
Web addresses of some representative internet
resources for protein identification from mass
spectrometry data
43
Mascot

• Among the first programs for identifying proteins
  by peptide mass fingerprinting, MOWSE, developed
  out of a collaboration between Imperial Cancer
  Research Fund (ICRF) and SERC Daresbury
  Laboratory, UK.
• The name chosen was an acronym of Molecular
  Weight Search. The MOWSE databases were fully
  indexed so as to allow very rapid searching and
  retrieval of sequence data. Subsequently, the
  software was further developed and renamed
  Mascot.
• Licensed and distributed by Matrix Science Ltd.
• Specialized tools include Peptide Mass
  Fingerprint, Sequence Query, and MS/MS Ion
  Search.
• Search output Web-based.
• Good visual representation of search quality
  (graphical probability chart).
• Simple graphical user interface.
• Reports MOWSE scores as a quantitative measure of
  search quality.

44
Mowse Scoring

• Rather than just counting the number of matching
  peptides, Mowse uses empirically determined
  factors to assign a statistical weight to each
  individual peptide match. Rapid identification
  of proteins by peptide-mass fingerprinting. Curr.
  Biol. 3327, 1993.)
• Scoring scheme assigns more weight to matches of
  higher molecular weight peptides (more
  discriminating).
• Compensates for the non-random distribution of
  fragment molecular weights in proteins of
  different sizes.
• Was first protein identification program to
  recognize that the relative abundance of peptides
  of a given length in a proteolytic digest depends
  on the lengths of both peptide and protein.
• Developed for MALDI peptide mass fingerprinting.
• Probability-Based Mowse
• Mascot incorporates a probability-based enhanced
  Mowse algorithm, described in Perkins et al.
  (Probability-based protein identification by
  searching sequence databases using mass
  spectrometry data. Electrophoresis 203551-3567,
  1999).
• A simple rule can be used to judge whether a
  result is significant or not. Different types of
  matching (peptide masses and fragment ions) can
  be combined in a single search.

45
Databases

• Three components are required for database
  searching support of proteomics MALDI or MS/MS
  data, the algorithms used to search protein
  databases with the MALDI or MS/MS data, and the
  protein databases themselves.
• The protein databases can be as small as one
  protein, can be large, public domain databases of
  all known and predicted proteins, or may be
  predicted open reading frames based on genomic
  sequence.
• A major challenge for database searching is that
  these protein databases are constantly changing,
  making database search results potentially
  obsolete as new entries are added that better fit
  the MALDI or MS data.
• Even as genomes are completed there is still flux
  as new coding regions are identified and novel
  mechanisms of increased translational complexity
  are better understood, such as alternative splice
  products, RNA editing, and ribosome slippage
  leading to novel, unexpected translation products.

46
Databases

• NCBI non-redundant (NCBInr)
• Non-redundant database from the National Center
  for Biotechnology Information for use with their
  search tools BLAST and Entrez comprised of
  translated sequences from the Genbank /EMBL/DDBJ
  consortium, SwissProt, Protein Information
  Resource (PIR), and Brookhaven Protein Data Bank
  (PDB).
• New releases are published bimonthly while
  updates occur daily.
• OWL
• OWL is comprised of Swiss-Prot, PIR, translated
  Genbank, and NRL-3D (PDB). All sequences are
  compared to Swiss-Prot to remove identical and
  trivially different sequences. Has not been
  updated since May, 1999.
• SWISSPROT
• While SwissProt contains only a subset of
  proteins, the proteins in this database are much
  better annotated and the sequences are much more
  reliable than those available in any other
  database.
• MSDB
• Comprehensive, non-identical protein sequence
  database maintained by the Proteomics Department
  at the Hammersmith Campus of Imperial College
  London. Designed specifically for MS
  applications.

47
Databases

• EST Clusters (dBEST)
• Division of GenBank that contains "single-pass"
  cDNA sequences, or Expressed Sequence Tags
  (ESTs), from a number of organisms.
• ESTs are relatively short, usually 3 end
  sequences from isolated mRNA.
• ESTs tend to be highly redundant and the
  sequence is much lower quality than from other
  sources. An advantage to using these ESTs is
  that they represent only expressed sequences (no
  introns) and include alternative splice variants
  their length, redundancy, and low quality are far
  improved by using clustered ESTs, such as the
  Compugen clusters.
• The EST database has some redundancy because it
  contains all possible combinations of alternative
  splice products, and so it can be very large (and
  slow to search).
• During a Mascot search, the nucleic acid
  sequences are translated in all six reading
  frames. dbEST is a very large database, and is
  divided into three sections EST_human,
  EST_mouse, and EST_others. Even so, searches of
  these databases take far longer than a search of
  one of the non-redundant protein databases. You
  should only search an EST database if a search of
  a protein database has failed to find a match.

48
MALDI-MS peptide fingerprint(tryptic digest of a
single protein)
1247.70
all peaks are (MH)
1116.67
1375.76
trypsin autolysis
1505.77
1424.85
1665.89
2005.07
1287.73
2719.48
1574.20

1811.85
1849.12
2476.21
2550.52

1000
1500
2000
2500
3000
m/z
49
Mascot (Matrix Science) for peptide mass
fingerprints
enter peak list
50
Mascot (Matrix Science) for peptide mass
fingerprints
possible identification
51
Mascot (Matrix Science) for peptide mass
fingerprints
get more info on probable proteins
list of all possible matches
52
Mascot (Matrix Science) for peptide mass
fingerprints
53
Mascot (Matrix Science) for peptide mass
fingerprints
tryptic peptides that matched
peptides that did not match
54
Mascot (Matrix Science) for peptide mass
fingerprints
tryptic peptides in protein sequence
better mass accuracy improves identification
process
55
LC-MS/MS for protein identification

• To provide further confirmation of the
  identification, if a tandem mass spectrometer
  (MS/MS) is available, peptide ions can be
  dissociated in the mass spectrometer to provide
  direct sequence information. Product ions from
  an MS/MS spectrum can be compared to available
  sequences using powerful software toolsl.
• For a single sample, LC-MS/MS analysis included
  two discrete steps (a) LC-MS peptide mapping to
  identify peptide ions from the digestion mixture
  and to deduce their molecular weights, and (b)
  LC-MS/MS of the previously detected peptides to
  obtain sequence information for protein
  identification.

56
Automated LC-MS/MS and database searching

• Current mass spectral technology permits the
  generation of MS/MS data at an unprecedented
  rate. Prior to the generation of powerful
  computer-based database searching strategies, the
  largest bottleneck in protein identification was
  the manual interpretation of this MS/MS data to
  extract the sequence information. Today, many
  computer-based search strategies that employ
  MS/MS data require no operator interpretation at
  all.
• Analogous to the approach described for peptide
  fingerprinting, these programs take the
  individual protein entries in a database and
  electronically "digest" them to generate a list
  of theoretical peptides for each protein.
• However, in the use of MS/MS data, these
  theoretical peptides are further manipulated to
  generate a second level of lists which contain
  theoretical fragment ion masses that would be
  generated in the MS/MS experiment for each
  theoretical peptide.

57
Automated LC-MS/MS and database searching

• These programs simply compare the list of
  experimentally determined fragment ion masses
  from the MS/MS experiment of the peptide of
  interest with the theoretical fragment ion masses
  generated by the computer program.
• The recent advent of data-dependant scanning
  functions has permitted the unattended
  acquisition of MS/MS data. An example of a raw
  MS/MS data searching program that takes
  particular advantage of this ability is SEQUEST.
• SEQUEST will input the data from a data-dependant
  LC/MS chromatogram and automatically strip out
  all of the MS/MS information for each individual
  peak, and submit it for database searching using
  the strategy discussed above.
• Each peak is treated as a separate data file,
  making it especially useful for the on-line
  separation and identification of individual
  components in a protein mixture.
• SEQUEST cross-correlates uninterpreted MS/MS mass
  spectra of peptides from protein/nucleotide
  databases. The software can analyze a single
  spectrum or an entire LC-MS/MS peptide map.
• No user interpretation of MS/MS spectra is
  involved.

58
Match?
Proteolytic Digest
MS/MS
Experimental Fragment Masses
100
41.63
HPLC-MS
475.3
100
588.3
456.7
54.28
325.2
50
60.16
50
701.3
815.4
62.59
49.20
38.27
46.75
410.3
33.59
851.5
29.02
212.1
912.5
0
900
1000
25
30
35
40
45
50
55
60
m/z
Time (min)
59
Direct identification of proteins using mass
spectrometry

• Removes the requirement to separate proteins by
  electrophoresis, etc
• MudPIT multidimensional protein identification
  technology, or Shotgun approach
• Protein lysate is digested with trypsin
• The peptide mixture is loaded onto a strong
  cation exchange (SCX) column (to separate on the
  basis of charge). A discrete fraction of peptides
  is displaced from the SCX column using a salt
  step gradient to a reversed-phase (RP) column (to
  separate on the basis of hydrophobicity).
• This fraction is eluted from the RP column into
  the MS. This iterative process is repeated,
  obtaining the fragmentation patterns of peptides
  in the original peptide mixture.
• MS/MS spectra are used to identify the proteins
  in the original protein complex.

Link et al. Nature Biotechnology 17, 676 (1999)
60
Large-scale analysis of the yeast proteome by
MudPIT

• Yates and coworkers, Nature Biotech. (2001) 19,
  242-247
• Assigned 5,540 peptides to MS spectra leading to
  the identification of 1,484 proteins from the S.
  cerevisiae proteome
• Of 6,216 ORFs in yeast genome, 83 have CAI
  values between 0 and 0.20 (i.e., predicted to be
  present at low levels) (Fig. A)
• MudPIT data 791 or 53.3 of the proteins
  identified have a CAI of lt0.2 (1.7 peptides per
  protein) (Fig. B)
• Number of peptides per protein increases with
  increasing CAI (Fig. C)

61
Approaches for Protein Sequencing and
Identification
Top Down
MS/MS
MIRERICACVLALGMLTGFTHAFGSKDAAADGKPLVVTTIGMIADAVKNI
AQGDVHLKGLMGPGVDPHLYTATAGDVEWLGNADLILYNGLHLETKMGEV
FSKLRGSRLVVAVSETIPVSQRLSLEEAEFDPHVWFDVKLWSYSVKAVYE
SLCKLLPGKTREFTQRYQAYQQQLDKLDAYVRRKAQSLPAERRVLVTAHD
AFGYFSRAYGFEVKGLQGVSTASEASAHDMQELAAFIAQRKLPAIFIESS
IPHKNVEALRDAVQARGHVVQIGGELFSDAMGDAGTSEGTYVGMVTHNID
TIVAALAR
The molecular mass of an intact protein defines
the native covalent state of a genes product,
including the effects of post-transcriptional/tran
slational modifications, and associated
heterogeneity, that are modulated by the actions
of other gene products . Moreover, the
fragmentation pattern from large proteins can
generate sufficient information for
identification from sequence databases,
particularly when combined with accurate mass
measurements of both the intact molecule and its
product ions.
62
In-Source Decay (ISD) for Protein Sequencing

• Peptides and large proteins can be fragmented by
  ISD
• Fragmentation occurs in the MALDI ion source
• not generally well controlled
• Reflectron TOF not necessary (linear TOF
  sufficient to measure product ions

• Complete sequence information not present, but
  extensive stretches of sequence from the N-
  and/or C-termini observed

cut
...TIDE...
63
MALDI-ISD-TOF Mass Spectrometry of Proteins
Sequence Information (In-Source Decay)
Protein 1
Molecular Weight Information
Protein 2
5000
10000
15000
20000
25000
m/z
64
MALDI-ISD-TOF Mass Spectrometry of Proteins

• ISD generally yields c-ions or z-ions

Sequence information from the N-terminus
A
L
Y
G
G
E
G
F
E
D
H
R
L
K/Q
E
D
PW?