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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

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proteomics tools
Proteomics Tools
  • Molecular Biology Tools
  • Separation & Display Tools
  • Protein Identification Tools
  • Protein Structure Tools
mass spectrometry needs
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
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
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
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.

How does a mass spectrometer work?

Create ions

Separate ions

Detect ions

  • Mass spectrum
  • Database analysis
slide7

Artificially trypsinated

Fragmented using trypsin

Artificial spectra built

Spot removed from gel

Generalized Protein Identification by MS

Spectrum of fragments generated

MATCH

Library

Database of sequences

(i.e. SwissProt)

ms principles
MS Principles
  • Different elements can be uniquely identified by their mass
ms principles10

N

-CH2-

OH

COOH

HO

-CH2CH-NH2

HO

HO

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

mass spectrometry
Mass Spectrometry
  • Analytical method to measure the molecular or atomic weight of samples
slide12

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.

mass spectrometry13
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
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 history15
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 principles16
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
Mass Spec Principles

Sample

+

_

Detector

Ionizer

Mass Analyzer

how does a mass spectrometer work18
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.

How does a mass spectrometer work?

Create ions

Separate ions

Detect ions

  • Mass spectrum
  • Database analysis
mass spectrometers
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)
typical mass spectrum
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)
m z ratio

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 spectrum24
Typical Mass Spectrum

Relative Abundance

aspirin

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

resolution resolving power

DM

M

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

resolution in ms27
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

Ion

Source

Mass

Filter

Inlet

Data

System

Detector

Sample Plate

Target

HPLC

GC

Solids probe

TOF

Quadrupole

Ion Trap

Mag. Sector

FTMS

Microch plate

Electron Mult.

Hybrid Detec.

PC’s

UNIX

Mac

MALDI

ESI

IonSpray

FAB

LSIMS

EI/CI

Mass Spectrometer Schematic
different ionization methods
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
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 ch 3 oh
EI Fragmentation of CH3OH

CH3OH

CH3OH+

CH3OH

CH2O=H+

+ H

CH3OH

+ CH3

+ OH

CH2O=H+

+ H

CHO=H+

Why wouldn’t Electron Impact be suitable

for analyzing proteins?

why you can t use ei 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 sub-fragments 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
Soft Ionization Methods

337 nm UV laser

Fluid (no salt)

+

_

Gold tip needle

cyano-hydroxy

cinnamic acid

MALDI

ESI

soft ionization
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
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
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)
electrospray ionization40
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
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
Matrix-Assisted Laser Desorption Ionization

337 nm UV laser

cyano-hydroxy

cinnamic acid

MALDI

maldi
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)
maldi ionization
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

+

+

+

-

+

+

-

-

+

-

+

+

+

+

+

+

maldi46
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
maldi seldi
MALDI =SELDI

337 nm UV laser

cyano-hydroxy

cinnaminic acid

MALDI

mass spectrometer schematic51

Turbo pumps

Diffusion pumps

Rough pumps

Rotary pumps

High Vacuum System

Ion

Source

Mass

Filter

Inlet

Data

System

Detector

Sample Plate

Target

HPLC

GC

Solids probe

TOF

Quadrupole

Ion Trap

Mag. Sector

FTMS

Microch plate

Electron Mult.

Hybrid Detec.

PC’s

UNIX

Mac

MALDI

ESI

IonSpray

FAB

LSIMS

EI/CI

Mass Spectrometer Schematic
different mass analyzers
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
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 ms54
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
quadrupole mass 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
q tof mass analyzer

NANOSPRAY

TIP

MCP

DETECTOR

PUSHER

HEXAPOLE

HEXAPOLE

COLLISION

CELL

TOF

QUADRUPOLE

REFLECTRON

SKIMMER

ION

SOURCE

HEXAPOLE

Q-TOF Mass Analyzer
mass spec equation tof
Mass Spec Equation (TOF)

2Vt2

m

=

z

L2

m= mass of ionL= drift tube length

z= charge of iont= time of travel

V= voltage

ion trap mass analyzer
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
ft icr fourier transform ion cyclotron resonance
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
current mass spec technologies
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
slide64

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

peptide mass fingerprinting
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
Principles of Fingerprinting

SequenceMass (M+H)Tryptic Fragments

>Protein 1

acedfhsakdfqea

sdfpkivtmeeewe

ndadnfekqwfe

>Protein 2

acekdfhsadfqea

sdfpkivtmeeewe

nkdadnfeqwfe

>Protein 3

acedfhsadfqeka

sdfpkivtmeeewe

ndakdnfeqwfe

acedfhsak

dfgeasdfpk

ivtmeeewendadnfek

gwfe

acek

dfhsadfgeasdfpk

ivtmeeewenk

dadnfeqwfe

acedfhsadfgek

asdfpk

ivtmeeewendak

dnfegwfe

4842.05

4842.05

4842.05

principles of fingerprinting69
Principles of Fingerprinting

SequenceMass (M+H)Mass Spectrum

>Protein 1

acedfhsakdfqea

sdfpkivtmeeewe

ndadnfekqwfe

>Protein 2

acekdfhsadfqea

sdfpkivtmeeewe

nkdadnfeqwfe

>Protein 3

acedfhsadfqeka

sdfpkivtmeeewe

ndakdnfeqwfe

4842.05

4842.05

4842.05

predicting peptide cleavages
Predicting Peptide Cleavages

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

slide71

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

protease cleavage rules
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
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
slide74

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

slide75

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

slide76

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
What Are Missed Cleavages?

SequenceTryptic Fragments (no missed cleavage)

>Protein 1

acedfhsakdfqea

sdfpkivtmeeewe

ndadnfekqwfe

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
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 12C-12

2H-2.014017780 amu 13C-13.00335, 14C-14.00324

masses in ms
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
Mass Calculation (Glycine)

NH2—CH2—COOH

Amino acid

R1—NH—CH2—CO—R3

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

amino acid residue masses
Amino Acid Residue Masses

Monoisotopic Mass

Glycine 57.02147

Alanine 71.03712

Serine 87.03203

Proline 97.05277

Valine 99.06842

Threonine 101.04768

Cysteine 103.00919

Isoleucine 113.08407

Leucine 113.08407

Asparagine 114.04293

Aspartic acid 115.02695

Glutamine 128.05858

Lysine 128.09497

Glutamic acid 129.0426

Methionine 131.04049

Histidine 137.05891

Phenylalanine 147.06842

Arginine 156.10112

Tyrosine 163.06333

Tryptophan 186.07932

amino acid residue masses82
Amino Acid Residue Masses

Average Mass

Glycine 57.0520

Alanine 71.0788

Serine 87.0782

Proline 97.1167

Valine 99.1326

Threonine 101.1051

Cysteine 103.1448

Isoleucine 113.1595

Leucine 113.1595

Asparagine 114.1039

Aspartic acid 115.0886

Glutamine 128.1308

Lysine 128.1742

Glutamic acid 129.1155

Methionine 131.1986

Histidine 137.1412

Phenylalanine 147.1766

Arginine 156.1876

Tyrosine 163.1760

Tryptophan 186.2133

preparing a peptide mass fingerprint database
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
Building A PMF Database

Sequence DBCalc.Tryptic Frags Mass List

>P12345

acedfhsakdfqea

sdfpkivtmeeewe

ndadnfekqwfe

>P21234

acekdfhsadfqea

sdfpkivtmeeewe

nkdadnfeqwfe

>P89212

acedfhsadfqeka

sdfpkivtmeeewe

ndakdnfeqwfe

acedfhsak

dfgeasdfpk

ivtmeeewendadnfek

gwfe

acek

dfhsadfgeasdfpk

ivtmeeewenk

dadnfeqwfe

acedfhsadfgek

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)

the fingerprint pmf algorithm
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
Query (MALDI) Spectrum

1007

1199

2211 (trp)

609

2098

450

1940 (trp)

698

500 1000 1500 2000 2500

query vs database
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

2098.9909

slide88

Database search

PeptIdent (ExPasy)

Mascot (Matrix Science)

MS-Fit (Prospector; UCSF)

ProFound (Proteometrics)

MOWSE (HGMP)

Human Genome Mapping Project

Mascot

theoretical

experimental

Protein ID

what you need to do pmf
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
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
mascot scoring
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)
slide98

-x

-e

P(x) = 1 - e

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

mascot mowse scoring
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
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
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
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
tandem mass spectrometry105
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
How Tandem MS sequencing works

Ser-Glu-Leu-Ile-Arg-Trp

  • 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…

Collision Cell

Ser-Glu-Leu-Ile-Arg

Ser-Glu-Leu-Ile

Ser-Glu-Leu

Etc…

slide109

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

slide110

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 proteomics111
Provides precise sequence-specific data

More informative than PMF methods (>90%)

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

MS-MS & Proteomics

AdvantagesDisadvantages

isotope coded affinity tag icat a quantitative method
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
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 *

how icat works

100

0

0

600

200

400

550

570

590

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

icat advantages vs disadvantages
ICATAdvantages vs. Disadvantages
  • 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
  • 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
mass spectrometer schematic118

Turbo pumps

Diffusion pumps

Rough pumps

Rotary pumps

High Vacuum System

Ion

Source

Mass

Filter

Inlet

Data

System

Detector

Sample Plate

Target

HPLC

GC

Solids probe

TOF

Quadrupole

Ion Trap

Mag. Sector

FTMS

Microch plate

Electron Mult.

Hybrid Detec.

PC’s

UNIX

Mac

MALDI

ESI

IonSpray

FAB

LSIMS

EI/CI

Mass Spectrometer Schematic
ms detectors
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
Mass Detectors

Electron Multiplier (Dynode)

slide121

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

slide122

Shotgun Proteomics: Multidimensional Protein

Identification Technology (MudPIT)

slide123

Characterization

MALDI-TOF MS

-(LC)-ESI-MS/MS

  • Identification
  • Post Translational modifications
  • Quantification

General Strategy for Proteomics Characterization

Fractionation &

Isolation

Liquid

Chromatography

2-DE

Peptides

Mass Spectrometry

Database Search

slide124

2D Chromatography

RP

SCX

Overview of Shotgun Proteomics: MudPIT

Protein Mixture

Digestion

Tandem Mass Spectrometer

Peptide Mixture

> 1,000 Proteins Identified

SEQUEST®

DTASelect & Contrast

MS/MS Spectrum

mudpit
MudPIT

IEX-HPLC

RP-HPLC

Trypsin

+ proteins

p53

slide126

2D Chromatography

RP

SCX

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

x 3~18

Acquiring MS/MS Datasets

Tandem MS Spectrum

Peptide Sequence is Inferred from Fragment ions

slide127

MS/MS of Peptide Mixtures

LC

MS

(MW Profile)

MS/MS

(AA Identity)

slide128

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

slide129

SEQUEST-PVM

Beowolf computing cluster

55 mixed CPU: Alpha chips and AMD

Athlon PC CPU

slide130

B

C

Control

A

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

slide131

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.
slide133

Purification

Cells/Tissues

Multiprotein Complex/

Organelle

Application of shotgun proteomics: Comprehensive Analysis of ComplexProtein Mixtures

Total Protein

Characterization

slide134

Yeast: A Perfect Model

  • Complete genome sequence information
  • An extensively studied organism
  • Optimal numbers of ORFs, easy for database search
slide135

Communication and Signal Transduction

Cell Growth, Division, DNA synthesis,

and Biogenesis

Energy

Ionic Homeostasis

Cell Rescue, Defense, Death, and Ageing

Transcription

Protein Destination

Protein Synthesis

Transport

Unclassified

Metabolism

Cellular Organization

Functional Categories of Yeast Proteins Identified

Used GO to determine functional groups

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

slide136

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

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

2-DE vs MudPIT
peptide masses from esi

m/z = (MW + nH+)

n

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)

peptide masses from esi139
Peptide Masses From ESI

Charge (n) is unknown, Key is to determine MW

Choose any two peaks separated by 1 charge

1301.4 = (MW + [n+1]H+)

1431.6 = (MW + nH+)

[n+1]

n

2 equations with 2 unknowns - solve for n first

n = 1300.4/130.2 = 10

Substitute 10 into first equation - solve for MW

MW = 14316 - (10x1.008) = 14305.9

14,305.14

esi transformation
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)
esi and protein structure
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
ESI and Protein Conformation

Native Azurin

Denatured Azurin

different ms ms modes
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
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