My contact details and information about submitting samples for MS

2 main types of MS are used for PROTEIN IDENTIFICATION PEPTIDE MASS FINGERPRINTING (PMF) –MALDI-ToF MS TANDEM MS (aka MSMS) –ELECTROSPRAY Q-ToF2

Proteins are chains of amino acids each of which have slightly different masses The protein chain can be cut selectively by sequence specific proteases at particular amino acids Trypsin cuts after lysine or arginine Protein digestion

The peptides produced have distinct weights These are accurately measured by mass spectrometry A list of these weights is like a fingerprint (a PEPTIDE MASS FINGERPRINT) This is unique to the protein and can be used to identify it = = = 89.3

Laser energy Peptide ions enter the Time of Flight tube separated on basis of mass Peptides co- crystallised with matrix Ionise peptides Detector mass/charge of every peptide peptide mass fingerprint MALDI-TOF-MS (Matrix Assisted Laser Desorption and Ionisation)

peptide mass spectrum a trypsin digest of a single protein every peak corresponds to the mass (m/z) of a peptide ion m / z = mass / charge relative intensity

Peptide mass spectrum Data converted to text List of peptide masses a peak list (pkl) = fingerprint

run digested protein on MS Database searches with PeptideMassFingerprint data sequence databases theoretical trypsin digest of every predicted protein list of calculated peptide masses compare identification made if match is found list of measured peptide masses “fingerprint”

peptide mass fingerprinting rapid high throughput large scale identification of proteins from organisms with completely sequenced genomes good tool for a first look at a sample BUT……. peptide mass fingerprinting will not always give an identification genome is not completely sequenced the full length protein sequence is not in the database modifications are present more than one protein is present in the sample alternative method of analysis – LC-MSMS

Samples in solution Compatible with HPLC Complex protein mixtures  Determine peptide masses  Peptide fragmentation  Peptide sequence ElectroSpray Ionisation (ESI) Mass Spectrometry on the Q-ToF2

MS2MS1 MS on the Q-ToF2

Tandem MS - peptide fragmentation series of peptide fragments each fragment is one amino acid longer than the next the series of fragments corresponds to the sequence of the peptide low energy collision fragments the peptide one fragmentation per peptide molecule cleavage usually occurs at the amide bond i.e. between residues

peptide fragmentation the series of fragments corresponds to the sequence of the peptide

Survey Mass Spectrum (MS) - intact peptides detected in a 1 second survey scan MSMS On-line LC-MSMS on the Q-ToF2 : peptides from a single protein Fragment Mass spectrum (MSMS) fragments from one peptide MSMS Many peptides are fragmented during a 60 minute run LC-MSMS generates much more data than fingerprinting mass of intact peptides & the fragment masses Search databases with much more data per protein

MSMS ions search data peak list (pkl) Peptide mass : charge state : intensity fragment mass : intensity : :

Mascot Search Overview Mascot is a search engine which uses mass spectrometry data to identify proteins from primary sequence databases

MASCOT provides 3 different search methods Peptide Mass Fingerprint peptide mass values Sequence Query peptide mass data plus amino acid sequence/composition MS/MS Ion Search uninterpreted MS/MS data from one or more peptides

Cut-off score for significance is different for every search

Peptide score Expect value Number of matching peptides Protein score Protein name Different species

A match is made by correlating the observed and predicted peptide masses and their fragment ion masses – the peptides themselves have not actually been sequenced Predicted mass and predicted pI Sequence coverage

All these proteins are hit #1 All have the same score and the same peptide masses match The order of the list within each hit, is meaningless i.e. cow is “top” here, but the sample is mouse

Click on the MS/MS Ions Search tool page INSTRUCTIONS FOR THE PRACTICAL COPY the 4 MSMS files onto the desktop

Standard search Input your name & your Use these standard defaults swissprot trypsin, 1 missed cleavage variable on Carbamidomethyl C variable on Oxidation M peptide charge +2, +3, +4 Browse to add an MSMS file to the search page Micromass PKL ESI-QUAD-TOF

Vary some parameters in subsequent searches try NCBInr and swissprot databases for MSMS3 add in variable phosphorylations for MSMS4 semi-trypsin for MSMS2 alter mass tolerances compare results with standard search

links MASCOT video tutorials Mass Spectrometry and Biotechnology Resource – lots of useful info – tutorials on de novo sequencing etc proteomics special interest group at NIH, includes archived videocasts of research seminars informatics tools e.g. peptidemass predicted digestion fragment tool British Society for Proteome Research British Mass Spectrometry society The Plasma Proteome Institute in Washington D.C. Unimod : protein modifications for mass spectrometry

De novo sequencing Sequence reads in N to C direction - PSGASTGVHEAMR

Example of good quality peptide match

Number of contiguous residues should be 5 or more Have 8 for this peptide – good quality match

Example of poor quality peptide

Longest stretch of contiguous reside calls is 2 – insufficient for good ID If this was the only peptide match it would be rejected by the user