Presentation on theme: "My contact details and information about submitting samples for MS http://www.nottingham.ac.uk/Biosciences/People/susan.liddell."— Presentation transcript:
1My contact details and information about submitting samples for MS
2We use 2 types of MS for PROTEIN IDENTIFICATION PEPTIDE MASS FINGERPRINTING (PMF)MALDI-ToF MSTANDEM MS (aka MSMS)ELECTROSPRAY Q-ToF2
3Protein digestion Proteins are chains of amino acids each of which have slightly different massesThe protein chain can be cut selectively bysequence specific proteases at particular amino acidsTrypsin cuts after lysine or arginine
4The peptides produced have distinct weights These are accurately measured by mass spectrometry89.3= 89.3A list of these weights is like a fingerprint(a PEPTIDE MASS FINGERPRINT)This is unique to the protein and can be used to identify it95.489.3112.1105.3= 402.295.489.3112.1105.397.1101.8= 601
5MALDI-TOF-MS (Matrix Assisted Laser Desorption and Ionisation) energyPeptide ions enter the Time of Flight tubeseparated on basis of massPeptides co-crystallised with matrixIonise peptidesSchematic of the maldiIn the basic process of MALDI analysisSample is prepared in a molar excess (~1000:1) of a matrix compoundThe Maldi plate is placed on the stageLaser is fired which excites molecules in the matrixSample and matrix molecules ejected together into the gas phase, yielding [M+H]+ ionsThe peptide ions are ejected into the TOF mass analyser, are reflected and then detectedThe ions are separated based on the principleconstant energy but different mass(At constant energy ions with low mass travel more slowly than ions with high mass)Smaller ions will reach the detector sooner than larger ionsThe flight time of each ion is recorded by the detector hence the name time of flight (TOF)From this information, the software calculates mass for each peptideFor each digested protein, a unique set of peptide masses will be produced – a unique identifier, like a human fingerprint.The data is used to interrogate the databases in the following wayDetectormass/charge of every peptidepeptide mass fingerprint
6peptide mass spectrum a trypsin digest of a single protein every peak corresponds to the mass (m/z) of a peptide ionm / z = mass / chargerelativeintensity
7= fingerprint Peptide mass spectrum Data converted to text a peak list (pkl).Data converted to textList of peptide massesEvery peak corresponds to the exact mass (m/z) of a peptide ionBecause of the specificity of the enzyme cutting only at known and predictable sequences, each protein gives rise to a set of peptides unique to that proteinFor each digested protein, a unique set of peptide masses will be produced – a unique identifier, like a human fingerprint.The data is used to interrogate the databases in the following way=fingerprint
8Database searches with PeptideMassFingerprint data run digested protein on MSsequence databasestheoretical trypsin digest of every predicted proteinlist of calculated peptide massescompareidentification made if match is foundFor an individual protein sample, the peptide mass data from the MALDI spectrum (shown here) is formatted as a text file.This experimental data is compared with the equivalent type of data from the databases, which is obtained by-taking the predicted protein sequences-performing a theoretical trypsin digestion (must be same enzyme as was used in your experimental digestion) and-calculating the masses of the peptides that would be expected to be producedComparison of the two will hopefully provide an identification of the proteinlist of measured peptide masses “fingerprint”.
9peptide mass fingerprinting rapidhigh throughputlarge scale identification of proteins from organisms whose genomes are completely sequencedgood tool for a first look at a sampleBUT…….peptide mass fingerprinting will not always give an identificationgenome is not completely sequencedthe full length protein sequence is not in the databasemodifications are presentmore than one protein is present in the samplealternative method of analysis - tandem MS
10ElectroSpray Ionisation (ESI) Mass Spectrometry on the Q-ToF2 Samples in solutionCompatible with HPLCComplex protein mixturesDetermine peptide massesPeptide fragmentationPeptide sequenceAnd this is based on elestrospray ionisation in which peptides are ionised by spraying a sample, in solution, through a charged inlet.A couple of different inlets are used to introduce samples - one is directly from individual single use needles, and the other one is a via a sprayer unit which is connected on-line to an HPLC system. As is shown here with our Q-ToF with the HPLC system (CapLC).The clear advantage of using HPLC is the separation of tryptic peptide mixtures prior to mass analysis. This allows more complex mixtures to be analysed.The other advantage of hooking up an HPLC is that sample delivery can be automatedQ-tof gathers more information than a standard MALDI, which provides additional peptide information, including the possibility of amino acid sequence.However, the run time for each sample on a Q-Tof analysis is longer (can be over an hour, for a standard protein spot) and is overall therefore not as high throughput a system as the MALDI.Also takes a lot more user intervention and nursing.
11MS on the Q-ToF2MS1MS2the Q-ToF is a hybrid instrument with more than one mass analyser which allows a type of analysis known as tandem ms (or MSMS)During tandem ms there are essentially two stages of mass analysis-what happens is that ions are delivered into the instrument and enter the first mass analyser the (MS1) (the quadrupole)here, particular peptide ions are selected for further analysis (this selection is governed by parameters that you set in the software)- only the selected ions are allowed to exit to the gas collision cell at any one time-In the gas cell, the selected peptides (called parent ions) are fragmented via low energy collision with molecules of Argon gas-the resultant fragments, the so-called daughter ions, are then swept into the second mass analyser (MS2), the time of flight tube where they are separated and detected-it is this 2 stage process which gives tandem MS it’s name-during a run, msms data can be recorded for several parent peptides at a time-so that even if several peptides co-elute at a particular point in the gradient, they should all be detected
12Tandem MS - peptide fragmentation low energy collision fragments the peptidecleavage usually occurs at the amide bond i.e. between residuesseries of peptide fragmentseach fragment is one amino acid longer than the nextthe series of fragments corresponds to the sequence of the peptideDuring low energy collisions in the Q-Tof, the most facile cleavage of the peptide occurs at the amide bond which leads predominantly to the formation of y type ions (retention of the charge at the C-terminal side) and some low molecular weight b type ions (retention of the charge at the N-terminal side).The range of different fragment ion masses present in the MS/MS spectrum may then be attributed to part or occasionally all of the peptide sequence.each peptide fragment in a series differs from its neighbour by one amino acid.possible to determine the amino-acid sequence by considering the mass difference between neighbouring peaks in a seriesHowever, the difficulty lies in the fact that the information in tandem-MS spectra is often not complete and that intervening peaks, which might or might not belong to the series, can confuse the analysis. For example, a mass difference of 114 Da might be found between two large peaks, but a very small peak might also be found at 57 Da between these two large peaks. This part of the spectrum could therefore correspond to one asparagine (residue mass = 114 Da) or two glycines (residue mass = 57 Da). In practice, experts can correctly interpret at least parts of tandem-MS spectra, whereas computer algorithms are, as yet, unreliable for determining amino-acid sequences. In
13peptide fragmentation How does amino acid sequencing on a mass spec work? Again the msms procedure is usedThe process depends on the low energy collisions in the gas cell being set such that each parent peptide ion is subject to only one hit and therefore only one cleavage eventThis cleavage most frequently occurs at a peptide bond in the backbone of the parent peptide chainSo from each group of parent peptides of a selected mass, a “nested” set of fragments are producedThe mass difference between 2 adjacent fragments (daughter fragments) corresponds to the residue that differs between themSo the sequence of peptide can be determined by working along the ion series spelling out each amino-acid in the chainThis is done with the assistance of software but even with this, depending on the quality of the spectrum produced, can take significant amounts of user timeThe resulting amino-acid sequence can then be used in a database search e.g. BLAST to try and identify the proteinThe spectrum here shows the daughter ions produced from one parent peptidein the nested set, the mass difference between 2 adjacent fragments (peptide ions/daughter fragments) corresponds to the residue that differs between the two fragmentsSo the sequence of peptide can be determined by working along the ion series spelling out each amino-acid in the chain.The resulting amino-acid sequence can then be used in a database search e.g. BLAST to try and identify the proteinEven when the quality of the spectrum is poor, it is often possible to pick out clean peaks, and read off residues of sequence – 3 is enough, combined with other information (e.g. mass data of peptide and parent) to form a good searchthe series of fragments corresponds to the sequence of the peptide
14De novo sequencingSequence reads in N to C direction - PSGASTGVHEAMR
15Many peptides are fragmented during a 60 minute run On-line LC-MSMS on the Q-ToF2 : peptides from a single proteinSurvey Mass Spectrum (MS) - intact peptidesdetected in a 1 second survey scanFragment Mass spectrum (MSMS)fragments from one peptideMSMSMSMSMSMSMany peptides are fragmented during a 60 minute runLC-MSMS generates much more data than fingerprintingmass of intact peptides & the fragment massesSearch databases with much more data per proteinMSMSMS/MS instruments can select a single peptide ion out of a spectrum by MS1This peptide ion is fragmented by collision with inert gasThe mass of the fragment ions is measured by MS2 = ToF
16MSMS ions search data peak list (pkl) 920.9598 241.0128 2 :MSMS ions search datapeak list (pkl)Peptide mass : charge state : intensityfragment mass : intensity
17Examples of open access search tools MASCOT3 types of searchesAldentePMF search toolPhenyxan MS data analysis platformidentification and characterization of proteins & peptides from mass spectrometry data
18Mascot Search Overview Mascot is a search engine which uses mass spectrometry data to identify proteins from primary sequence databases
19MASCOT provides 3 different search methods Peptide Mass Fingerprintpeptide mass valuesSequence Querypeptide mass data plus amino acid sequence/compositionMS/MS Ion Searchuninterpreted MS/MS data from one or more peptides
20Cut-off score for significance is different for every search Decoy database searchCut-off score for significance is different for every search
21Number of matching peptides Protein score Protein name Peptide scoreExpect valueProtein Hit ListThe body of the Peptide Summary report contains a tabular listing of the proteins, sorted by descending protein score. For each protein, the first line contains the accession string, (linked to the corresponding Protein View), the protein molecular mass, and the protein score. The number of queries matched to the protein completes the first line. The second line is the protein description taken from the Fasta entry. This is followed by a table summarising the matched peptide masses.The table columns contain:Query number, hyperlinked to Peptide View.Experimental m/z valueExperimental m/z transformed to a relative molecular massRelative molecular mass calculated from the matched peptide sequenceDifference (error) between the experimental and calculated massesNumber of missed cleavage sitesIons score - If there are duplicate matches to the same peptide, then the lower scoring matches are shown in brackets.Expectation value for the peptide match. (The number of times we would expect to obtain an equal or higher score, purely by chance. The lower this value, the more significant the result).Rank of the ions match, (1 to 10, where 1 is the best match).A letter U if the peptide sequence is unique to the protein hitSequence of the peptide in 1-letter code. The residues that bracket the peptide sequence in the protein are also shown, delimited by periods. If the peptide forms the protein terminus, then a dash is shown instead.Any variable modifications found in the peptideDifferent species
22Predicted mass and predicted pI Sequence coverageOnly the peptide masses and their fragment ion masses are matched– the peptides themselves have not actually been sequenced
23All these proteins are hit #1 All have the same score and the same peptide masses matchThe order of the list within each hit, is meaninglessi.e. cow is “top” here, but the sample is mouse
24Download the MSMS files 1 to 4 onto the desktop Click on the MS/MS Ions Search tool page
25Standard search input your name & your e-mail use standard defaults swissprottrypsin, 1 missed cleavagevariable on Carbamidomethyl Cvariable on Oxidation Mpeptide charge +2, +3, +4Copy MSMS files to desktopBrowse to add file to search pageMicromass PKLESI-QUAD-TOF
26Vary some parameters in subsequent searches try NCBInr and swissprot databases for MSMS3add in variable phosphorylations for MSMS4semi-trypsinalter mass tolerancescompare results with standard search
27Selected references and reviews Gorg A, Weiss W, Dunn MJ.Current two-dimensional electrophoresis technology for proteomics.Proteomics Dec;4(12):Two-dimensional gel electrophoresis: an overview, PagesDavid E. GarfinTrends in Analytical ChemistryVolume 22, Issue 5, Pages (May 2003)The current state of two-dimensional electrophoresis with immobilized pH gradients.Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W.Electrophoresis Apr;21(6):ANALYSIS OF PROTEINS AND PROTEOMES BY MASS SPECTROMETRYMatthias Mann, Ronald C. Hendrickson, and Akhilesh PandeyAnnual Review of Biochemistry July 2001, Vol. 70, ppChallenges in mass spectrometry-based proteomics.Reinders J, Lewandrowski U, Moebius J, Wagner Y, Sickmann A.Proteomics Dec;4(12):Plant proteome analysis.Canovas FM, Dumas-Gaudot E, Recorbet G, Jorrin J, Mock HP, Rossignol M.Proteomics Feb;4(2):Subcellular proteomics.Dreger M.Mass Spectrom Rev Jan-Feb;22(1):27-56.Functional organization of the yeast proteome by systematic analysis of protein complexes.Gavin AC, Bosche M, et alNature Jan 10;415(6868):141-7.
28Development Feb;131(3):Drosophila ventral furrow morphogenesis: a proteomic analysis.Lei Gong, Mamta Puri, Mustafa Ünlü, Margaret Young, Katherine Robertson, Surya Viswanathan, Arun Krishnaswamy, Susan R. Dowd and Jonathan S. MindenState-of-the-art in phosphoproteomicsProteomics 2005 Early View i.e. find it on the journals early view section of the web siteJoerg Reinders, Albert SickmannGlobal quantitative phosphoprotein analysis using Multiplexed Proteomics technology.Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF.Proteomics Jul;3(7):Steen H, Mann M.The ABC's (and XYZ's) of peptide sequencing.Nat Rev Mol Cell Biol Sep;5(9): Review.
29links http://www.swissproteomicsociety.org/digest Swiss Proteomics Society. The “digest” provides a consolidated selection of articles published in all scientific publications that are pertinent to proteomics – finds all the interesting and relevant papers for you!proteomics special interest group at NIH, includes archived videocasts of research seminarsproteome informatics tools e.g. peptidemass predicted digestion fragment toolBritish Society for Proteome ResearchBritish Mass Spectrometry societyThe Plasma Proteome Institute in Washington D.C.Unimod : protein modifications for mass spectrometryMass Spectrometry and Biotechnology Resource – lots of useful info – tutorials on de novo sequencing etc