MS - eLisa UGM

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Transcript MS - eLisa UGM

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
MATC
H
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
L-dopa
N -CH2OH
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-  NaClNaCl  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
Triple Quadrapole • Database
analysis
• AA seq
– MALDI-QqTOF
• AA seq and MW
– QqTOF
• AA seq and
protein modif.
Mass spectrometers
Linear Time Of Flight tube
ion source
•
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
Reflector Time Offrom
Flight tube
multiple laser bursts are averaged. Multiple laser
detector
time of flight
ion source
•
•
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
detector
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)
reflector
time of flight
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 ion’s 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:
DM
M
M is the mass number of the observed mass
(DM) 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
PC’s
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 wouldn’t Electron Impact be suitable
for analyzing proteins?
Why You Can’t 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 10,000’s 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
1960’s by Malcolm Dole but put into
practice in 1980’s 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.
•
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.
Electrospray Ionization
• Sample dissolved in polar, volatile buffer
(no salts) and pumped through a stainless
steel capillary (70 - 150 mm) at a rate of 10100 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)
Electrospray (Detail)
Electrospray Ionization
• Can be modified to “nanospray” system
with flow < 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)
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
peptide mixture
embedded in
light absorbing
chemicals (matrix)
pulsed
UV or IR laser
(3-4 ns)
vacuum
+ +
+
+
+ + +
+
strong
electric
field
Vacc
cloud of
protonated
peptide molecules
+
detector
+
Time Of Flight tube
Principal for MALDI-TOF MASS
Linear Time Of Flight tube
ion source
detector
time of flight
Reflector Time Of Flight tube
ion source
detector
reflector
time of flight
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
PC’s
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)
– Highest resolution, exact mass, costly
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 ID’s
by mass
• LC-MS - Liquid Chromatography MS
– separates delicate compounds in HPLC column and
ID’s by mass
• MS-MS - Tandem Mass Spectrometry
– separates compound fragments by magnetic field and
ID’s by mass
• LC/LC-MS/MS-Tandem LC and Tandem MS
– Separates by HPLC, ID’s by mass and AA sequence
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
HEXAPOLE
COLLISION
CELL
TOF
REFLECTRON
SKIMMER
HEXAPOLE
Mass Spec Equation (TOF)
2Vt2
m
=
z
L2
m = mass of ion
z = charge of ion
V = voltage
L = drift tube length
t = time of travel
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
(trypsin)
Study protein
complexes
without gel
electrophoresis
Complex mixture is
simplified prior to
MS/MS by 2D LC
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
Mass (M+H)
>Protein 1
acedfhsakdfqea
sdfpkivtmeeewe
ndadnfekqwfe
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
XX[FYW]--[!P]XXX
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
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
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
1233.7
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
2526.4
2542.4
3329.6
4211.4
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
1H-1.007828503
amu
2H-2.014017780 amu
12C-12
13C-13.00335, 14C-14.00324
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)
NH2—CH2—COOH
Amino acid
R1—NH—CH2—CO—R3
Residue
Monoisotopic Mass
1H = 1.007825
12C = 12.00000
14N = 14.00307
16O = 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
Alanine
Serine
Proline
Valine
Threonine
Cysteine
Isoleucine
Leucine
Asparagine
57.02147
71.03712
87.03203
97.05277
99.06842
101.04768
103.00919
113.08407
113.08407
114.04293
Aspartic acid
Glutamine
Lysine
Glutamic acid
Methionine
Histidine
Phenylalanine
Arginine
Tyrosine
Tryptophan
115.02695
128.05858
128.09497
129.0426
131.04049
137.05891
147.06842
156.10112
163.06333
186.07932
Amino Acid Residue Masses
Average Mass
Glycine
Alanine
Serine
Proline
Valine
Threonine
Cysteine
Isoleucine
Leucine
Asparagine
57.0520
71.0788
87.0782
97.1167
99.1326
101.1051
103.1448
113.1595
113.1595
114.1039
Aspartic acid
Glutamine
Lysine
Glutamic acid
Methionine
Histidine
Phenylalanine
Arginine
Tyrosine
Tryptophan
115.0886
128.1308
128.1742
129.1155
131.1986
137.1412
147.1766
156.1876
163.1760
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
Calc. Tryptic Frags
>P12345
acedfhsakdfqea
sdfpkivtmeeewe
ndadnfekqwfe
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)
• Doesn’t require too much sample optimization
• Can be done by a moderately skilled operator (don’t
need to be an MS expert)
• Widely supported by web servers
• Improves as DB’s 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
Ser-Glu-Leu-Ile-Arg-Trp
Collision Cell
• Fragmentation of the peptides occur
in a predictable fashion, mainly at the
peptide bonds
Ser-Glu-Leu-Ile-Arg
• The resulting daughter ions have
masses that are consistent with
known molecular weights of
dipeptides, tripeptides,
tetrapeptides…
Ser-Glu-Leu
Ser-Glu-Leu-Ile
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
Biotin Affinity tag:
Binds tightly to
streptavidin-agarose
resin
Reactive group: Thiol-reactive
group will bind to Cys
O
Linker: Heavy version will
have deuteriums at *
Light version will have
hydrogens at *
NH
NH
H
N
*
S
O
*
O
O
*
O
*
H
N
I
O
The ICAT Reagent
How ICAT works?
Affinity isolation
on streptavidin
beads
Lyse &
Label
Quantification
MS
Identification
MS/MS
NH2-EACDPLRCOOH
Light
100
100
MIX
Heavy
Proteolysis
(ie trypsin)
0
0
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
• Slight chromatography
differences
• Tag fragmentation
• Cys-specific label reduces
sample complexity
• Peptides can be
sequenced directly if
tandem MS-MS is used
• 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
PC’s
UNIX
Mac
MS Detectors
• Early detectors used photographic film
• Today’s 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
m-(LC)-ESI-MS/MS
Overview of Shotgun Proteomics: MudPIT
Protein Mixture
Digestion
Tandem Mass
Spectrometer
2D Chromatography
RP
Peptide
Mixture
SCX
> 1,000 Proteins
Identified
MS/MS Spectrum
PySpzS5609 #2438 RT: 66.03 AV: 1 NL: 8.37E6
T: + c d Full ms2 [email protected] [ 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
915.53
986.50
882.07
600.24
481.13
869.23
1258.56
1033.60
1312.35
1142.43
1123.49
1356.10
1195.44
0
200
300
400
500
600
700
800
900
m/z
1000
1100
1200
1300
1400
SEQUEST®
DTASelect &
Contrast
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 ms2 [email protected] [ 190.00-1470.00]
545.31
100
95
#1
CALCULATE #2
#3
#4
#5
…
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
427.27
559.13
651.14
408.74
399.24
217.91
1241.39
914.34
317.17
10
5
1032.43
895.33
30
K.TVLIMELINNVAK.K
L.NAKMELLIDLVKA.Q
E.ELAILMQNNIIGE.N
A.CGPSRQNLLNAMP.S
L.FAPLQEIINGILE.G
432.40
669.39
1027.22
915.53
986.50
882.07
600.24
481.13
869.23
1258.56
1033.60
1312.35
1142.43
1123.49
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
A
B
C
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,
hypothetical
Known,
biochem.
or genetics
MIPS
6368
1568
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+)
n
1301.4 = (MW + [n+1]H+)
[n+1]
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