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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 l.jpg
Proteomics Tools

  • Molecular Biology Tools

  • Separation & Display Tools

  • Protein Identification Tools

  • Protein Structure Tools


Mass spectrometry needs l.jpg
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 l.jpg
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 l.jpg
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 l.jpg

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 l.jpg

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 l.jpg
MS Principles

  • Different elements can be uniquely identified by their mass


Ms principles10 l.jpg

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 l.jpg
Mass Spectrometry

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


Slide12 l.jpg

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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
Mass Spec Principles

Sample

+

_

Detector

Ionizer

Mass Analyzer


How does a mass spectrometer work18 l.jpg

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 l.jpg
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 l.jpg
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 l.jpg

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 l.jpg
Typical Mass Spectrum

Relative Abundance

aspirin

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


Resolution resolving power l.jpg

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 l.jpg
Resolution in MS

783.455

QTOF

784.465

785.475

783.6


Mass spectrometer schematic l.jpg

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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
Soft Ionization Methods

337 nm UV laser

Fluid (no salt)

+

_

Gold tip needle

cyano-hydroxy

cinnamic acid

MALDI

ESI


Soft ionization l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
Matrix-Assisted Laser Desorption Ionization

337 nm UV laser

cyano-hydroxy

cinnamic acid

MALDI


Maldi l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
MALDI =SELDI

337 nm UV laser

cyano-hydroxy

cinnaminic acid

MALDI


Maldi seldi spectra l.jpg
MALDI/SELDI Spectra

Normal

Tumor


Mass spectrometer schematic51 l.jpg

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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg

NANOSPRAY

TIP

MCP

DETECTOR

PUSHER

HEXAPOLE

HEXAPOLE

COLLISION

CELL

TOF

QUADRUPOLE

REFLECTRON

SKIMMER

ION

SOURCE

HEXAPOLE

Q-TOF Mass Analyzer


Mass spec equation tof l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg

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 l.jpg
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 l.jpg
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 l.jpg
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 l.jpg
Predicting Peptide Cleavages

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


Slide71 l.jpg

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


Protease cleavage rules l.jpg
Protease Cleavage http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#TrypsRules

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 l.jpg
Why Trypsin?http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

  • 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 l.jpg

Digest with specific proteasehttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg

Digest with specific proteasehttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg

Digest with trypsinhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg
What Are Missed Cleavages?http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg
Calculating Peptide Masseshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

  • 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 l.jpg
Masses in MShttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

  • 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 l.jpg
Mass Calculation (Glycine)http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg
Amino Acid Residue Masseshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg
Amino Acid Residue Masseshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg
Preparing a Peptide Mass Fingerprint Databasehttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

  • 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 l.jpg
Building A PMF Databasehttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg
The Fingerprint (PMF) Algorithmhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

  • 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 l.jpg
Query (MALDI) Spectrumhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

1007

1199

2211 (trp)

609

2098

450

1940 (trp)

698

500 1000 1500 2000 2500


Query vs database l.jpg
Query vs. Databasehttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg

Database searchhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

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 l.jpg
What You Need To Do PMFhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

  • 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 l.jpg
PMF on the Webhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

  • 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


  • Profound l.jpg
    ProFoundhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Profound results l.jpg
    ProFound Resultshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Mowse l.jpg
    MOWSEhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Peptident l.jpg
    PeptIdenthttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Mascot l.jpg
    MASCOThttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Mascot scoring l.jpg
    Mascot Scoringhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg

    -xhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    -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


    Mascot99 l.jpg
    MASCOThttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Mascot mowse scoring l.jpg
    Mascot/Mowse Scoringhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg
    Advantages of PMFhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg
    Limitations With PMFhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg
    Tandem Mass Spectrometryhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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


    Ms ms proteomics l.jpg
    MS-MS & Proteomicshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Tandem mass spectrometry105 l.jpg
    Tandem Mass Spectrometryhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg
    How Tandem MShttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps 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 l.jpg

    Data Analysis Limitationshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    -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 l.jpg

    Advantages of Tandem Mass Spechttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg

    Provides precise sequence-specific datahttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg
    ISOTOPE-CODED AFFINITY TAG (ICAT): a quantitative methodhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg
    Example of an ICAT Reagenthttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 *


    The icat reagent l.jpg
    The ICAT Reagenthttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    How icat works l.jpg

    100http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 quantitation l.jpg
    ICAT Quantitationhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Icat advantages vs disadvantages l.jpg
    ICAThttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#TrypsAdvantages 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 l.jpg

    Turbo pumpshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg
    MS Detectorshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg
    Mass Detectorshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    Electron Multiplier (Dynode)


    Slide121 l.jpg

    Limitations of Proteomicshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    -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 l.jpg

    Shotgun Proteomics: http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#TrypsMultidimensional Protein

    Identification Technology (MudPIT)


    Slide123 l.jpg

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

    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 l.jpg

    2D Chromatographyhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg
    MudPIThttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    IEX-HPLC

    RP-HPLC

    Trypsin

    + proteins

    p53


    Slide126 l.jpg

    2D Chromatographyhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg

    MS/MS of Peptide Mixtureshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    LC

    MS

    (MW Profile)

    MS/MS

    (AA Identity)


    Slide128 l.jpg

    Matching MS/MS Spectra tohttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg

    SEQUEST-PVMhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    Beowolf computing cluster

    55 mixed CPU: Alpha chips and AMD

    Athlon PC CPU


    Slide130 l.jpg

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

    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 l.jpg

    Post Analysis Software http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#TrypsDTASelect: 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 l.jpg

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

    Cells/Tissues

    Multiprotein Complex/

    Organelle

    Application of shotgun proteomics: Comprehensive Analysis of ComplexProtein Mixtures

    Total Protein

    Characterization


    Slide134 l.jpg

    Yeast: A Perfect Modelhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • Complete genome sequence information

    • An extensively studied organism

    • Optimal numbers of ORFs, easy for database search


    Slide135 l.jpg

    Communication and Signal Transductionhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg

    Summary of MudPIThttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg

    Widely used, highly commercializedhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg

    m/zhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps = (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 l.jpg
    Peptide Masses From ESIhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    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 l.jpg
    ESI Transformationhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg
    Maximum Entropyhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


    Esi and protein structure l.jpg
    ESI and Protein Structurehttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg
    ESI and Protein Conformationhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    Native Azurin

    Denatured Azurin


    Different ms ms modes l.jpg
    Different MS-MS Modeshttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

    • 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 l.jpg

    THE ENDhttp://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps


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