Mass Spectrometry: Methods & Theory

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 Separate ions Detect ions Mass spectrum Database analysis Ionization method MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif.

Generalized Protein Identification by MS
Spot removed from gel Fragmented using trypsin Spectrum of fragments generated MATCH Library Artificial spectra built 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 Ethanol N OH HO -CH2- -CH2CH-NH2 COOH HO CH3CH2OH MW = MW = MW = 46.1

Mass Spectrometry Analytical method to measure the molecular or atomic weight of samples

Weighing proteins NaCl + e-  NaCl- NaCl  NaCl+ + e-
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 Spectrometry For small organic molecules the MW can be determined to within 5 ppm or % 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
Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize) 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 Detect ions Mass spectrum Database analysis Ionization method MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif.

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 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)

Molecular weight divided by the charge on this protein
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: M is the mass number of the observed mass (DM) is the difference between two masses that can be separated DM M

Resolution in MS

Resolution in MS QTOF 783.6

Mass Spectrometer Schematic
Inlet Ion Source Mass Filter Detector Data System High Vacuum System Turbo pumps Diffusion pumps Rough pumps Rotary pumps Sample Plate Target HPLC GC Solids probe MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. Hybrid Detec. PC’s UNIX Mac

Different Ionization Methods
Electron Impact (EI - Hard method) small molecules, 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
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 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
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 ( mm) at a rate of 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, 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)

HT Spotting on a MALDI Plate

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 + + + - + + - - + - + + + + + +

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

Principal for MALDI-TOF MASS

MALDI = SELDI 337 nm UV laser cyano-hydroxy cinnaminic acid MALDI

MALDI/SELDI Spectra Normal Tumor

Turbo pumps Diffusion pumps Rough pumps Rotary pumps High Vacuum System Inlet Ion Source Mass Filter Detector Data System Sample Plate Target HPLC GC Solids probe MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. Hybrid Detec. 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

Both separate by MW and AA seq
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 TOF QUADRUPOLE NANOSPRAY MCP TIP DETECTOR PUSHER
ION SOURCE HEXAPOLE COLLISION CELL QUADRUPOLE MCP DETECTOR REFLECTRON SKIMMER PUSHER

Mass Spec Equation (TOF)
2Vt2 = z L2 m = mass of ion L = drift tube length z = charge of ion t = time of travel V = voltage

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
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 Study protein complexes without gel electrophoresis
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

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 Mass (M+H) Tryptic Fragments >Protein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe >Protein 2 acekdfhsadfqea nkdadnfeqwfe >Protein 3 acedfhsadfqeka ndakdnfeqwfe acedfhsak dfgeasdfpk ivtmeeewendadnfek gwfe acek dfhsadfgeasdfpk ivtmeeewenk dadnfeqwfe acedfhsadfgek asdfpk ivtmeeewendak dnfegwfe

Principles of Fingerprinting
Sequence Mass (M+H) Mass Spectrum >Protein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe >Protein 2 acekdfhsadfqea nkdadnfeqwfe >Protein 3 acedfhsadfqeka ndakdnfeqwfe

Predicting Peptide Cleavages

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

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) , , , , , , ,

Digest with specific protease
546 aa 60 kDa; Da pI = 4.75 >RBME00320 Contig0311_ _ 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:

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 Tryptic Fragments (no missed cleavage) >Protein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe acedfhsak ( ) dfgeasdfpk ( ) ivtmeeewendadnfek ( ) gwfe ( ) Tryptic Fragments (1 missed cleavage) acedfhsak ( ) dfgeasdfpk ( ) ivtmeeewendadnfek ) gwfe ( ) acedfhsakdfgeasdfpk ( ) ivtmeeewendadnfekgwfe ( ) dfgeasdfpkivtmeeewendadnfek ( )

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 ( ) Add mass of H+ ( to get M+H) If Met is oxidized add If Cys has acrylamide adduct add If Cys is iodoacetylated add Other modifications are listed at 1H amu 12C-12 2H amu 13C , 14C

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 Glycine Amino Acid Mass 5xH + 2xC + 2xO + 1xN = amu Glycine Residue Mass 3xH + 2xC + 1xO + 1xN = amu Monoisotopic Mass 1H = 12C = 14N = 16O =

Amino Acid Residue Masses
Monoisotopic Mass Glycine Alanine Serine Proline Valine Threonine Cysteine Isoleucine Leucine Asparagine Aspartic acid Glutamine Lysine Glutamic acid Methionine Histidine Phenylalanine Arginine Tyrosine Tryptophan

Amino Acid Residue Masses
Average Mass Glycine Alanine Serine Proline Valine Threonine Cysteine Isoleucine Leucine Asparagine Aspartic acid Glutamine Lysine Glutamic acid Methionine Histidine Phenylalanine Arginine Tyrosine Tryptophan

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 Mass List >P12345 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe >P21234 acekdfhsadfqea nkdadnfeqwfe >P89212 acedfhsadfqeka ndakdnfeqwfe acedfhsak dfgeasdfpk ivtmeeewendadnfek gwfe acek dfhsadfgeasdfpk ivtmeeewenk dadnfeqwfe acedfhsadfgek asdfpk ivtmeeewendak dnfegwfe (P21234) (P12345) (P89212) (P12345) (P89212) (P12345) (P21234) (P21234) (P89212) (P89212) (P21234) (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

Query vs. Database Query Masses Database Mass List Results
(P21234) (P12345) (P89212) (P12345) (P89212) (P12345) (P21234) (P21234) (P89212) (P89212) (P21234) (P12345) 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 theoretical experimental Protein ID

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 = ± 0.1 Da) A PMF website (Prowl, ProFound, Mascot, etc.)

PMF on the Web ProFound MOWSE PeptideSearch Mascot PeptIdent
MOWSE PeptideSearch Mascot PeptIdent

ProFound

ProFound Results

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. P(x) = 1 - e -e -x Scores greater than 72 are significant

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 sent for further fragmentation

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 Fragmentation of the peptides occur in a predictable fashion, mainly at the peptide bonds The resulting daughter ions have masses that are consistent with known molecular weights of dipeptides, tripeptides, tetrapeptides… Ser-Glu-Leu-Ile-Arg-Trp Collision Cell Ser-Glu-Leu-Ile-Arg Ser-Glu-Leu-Ile Ser-Glu-Leu Etc…

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 Disadvantages
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

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 Linker: Heavy version will have deuteriums at * Light version will have hydrogens at *

The ICAT Reagent

Affinity isolation on streptavidin beads
How ICAT works? Affinity isolation on streptavidin beads Lyse & Label Quantification MS Identification MS/MS NH2-EACDPLR-COOH Light 100 200 400 600 100 550 570 590 MIX Heavy Proteolysis (ie trypsin) m/z m/z

ICAT Quantitation

ICAT Advantages vs. Disadvantages
Estimates relative protein levels between samples with a reasonable level of accuracy (within 10%) Can be used on complex mixtures of proteins Cys-specific label reduces sample complexity Peptides can be sequenced directly if tandem MS-MS is used Yield and non specificity Slight chromatography differences Expensive Tag fragmentation Meaning of relative quantification information No presence of cysteine residues or not accessible by ICAT reagent

Turbo pumps Diffusion pumps Rough pumps Rotary pumps High Vacuum System Inlet Ion Source Mass Filter Detector Data System Sample Plate Target HPLC GC Solids probe MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. Hybrid Detec. 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 Liquid Chromatography 2-DE Peptides Characterization Identification Post Translational modifications Quantification MALDI-TOF MS -(LC)-ESI-MS/MS Mass Spectrometry Database Search

Tandem Mass Spectrometer > 1,000 Proteins Identified
Overview of Shotgun Proteomics: MudPIT Protein Mixture Digestion Tandem Mass Spectrometer Peptide Mixture SCX RP 2D Chromatography > 1,000 Proteins Identified SEQUEST® DTASelect & Contrast MS/MS Spectrum

MudPIT IEX-HPLC RP-HPLC Trypsin + proteins p53

Peptide Sequence is Inferred from Fragment ions
Acquiring MS/MS Datasets SCX RP 2D Chromatography MudPIT Cycle load sample wash salt step RP gradient re-equilibration x 3~18 Tandem MS Spectrum Peptide Sequence is Inferred from Fragment ions

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 #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

SEQUEST-PVM Beowolf computing cluster
55 mixed CPU: Alpha chips and AMD Athlon PC CPU

20,000s of SEQUEST Output Files
Filtering, Assembling & Comparing Protein Lists 20,000s of SEQUEST Output Files Protein List PARSE ASSEMBLE DTASelect FILTER Criteria Sets Contrast COMPARE Summary Table Control A B C VISUALLY ASSESS SPECTRUM/PEPTIDE MATCHES

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.

Multiprotein Complex/
Application of shotgun proteomics: Comprehensive Analysis of Complex Protein Mixtures Purification Multiprotein Complex/ Organelle Cells/Tissues Total Protein Characterization

Yeast: A Perfect Model Complete genome sequence information
Database ORF Unknown, uncoding, hypothetical Known, biochem. or genetics MIPS 6368 1568 4344 YPD 6145 1833 4270 SGD ~6000 NA Complete genome sequence information An extensively studied organism Optimal numbers of ORFs, easy for database search

Used GO to determine functional groups
Functional Categories of Yeast Proteins Identified Used GO to determine functional groups Ionic Homeostasis Communication and Signal Transduction Cell Rescue, Defense, Death, and Ageing Energy Protein Destination Transcription Transport Protein Synthesis Cell Growth, Division, DNA synthesis, and Biogenesis Unclassified Metabolism Cellular Organization Washburn et al. Nature Biotechnology 19, (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 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

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 = (MW + nH+) = (MW + [n+1]H+) n [n+1] 2 equations with 2 unknowns - solve for n first n = /130.2 = 10 Substitute 10 into first equation - solve for MW MW = (10x1.008) = 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

Mass Spectrometry: Methods & Theory

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Mass Spectrometry: Methods & Theory

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