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Presentation on theme: "My contact details and information about submitting samples for MS http://www.nottingham.ac.uk/Biosciences/People/susan.liddell."— Presentation transcript:

1 My contact details and information about submitting samples for MS

2 We use 2 types of MS for PROTEIN IDENTIFICATION
PEPTIDE MASS FINGERPRINTING (PMF) MALDI-ToF MS TANDEM MS (aka MSMS) ELECTROSPRAY Q-ToF2

3 Protein digestion 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

4 The peptides produced have distinct weights
These are accurately measured by mass spectrometry 89.3 = 89.3 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 95.4 89.3 112.1 105.3 = 402.2 95.4 89.3 112.1 105.3 97.1 101.8 = 601

5 MALDI-TOF-MS (Matrix Assisted Laser Desorption and Ionisation)
energy Peptide ions enter the Time of Flight tube separated on basis of mass Peptides co-crystallised with matrix Ionise peptides Schematic of the maldi In the basic process of MALDI analysis Sample is prepared in a molar excess (~1000:1) of a matrix compound The Maldi plate is placed on the stage Laser is fired which excites molecules in the matrix Sample and matrix molecules ejected together into the gas phase, yielding [M+H]+ ions The peptide ions are ejected into the TOF mass analyser, are reflected and then detected The ions are separated based on the principle constant 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 ions The 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 peptide For 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 Detector mass/charge of every peptide peptide mass fingerprint

6 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

7 = fingerprint Peptide mass spectrum Data converted to text
a peak list (pkl) . Data converted to text List of peptide masses Every peak corresponds to the exact mass (m/z) of a peptide ion Because 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 protein For 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

8 Database searches with PeptideMassFingerprint data
run digested protein on MS sequence databases theoretical trypsin digest of every predicted protein list of calculated peptide masses compare identification made if match is found For 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 produced Comparison of the two will hopefully provide an identification of the protein list of measured peptide masses “fingerprint” .

9 peptide mass fingerprinting
rapid high throughput large scale identification of proteins from organisms whose genomes are completely sequenced 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 - tandem MS

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

11 MS on the Q-ToF2 MS1 MS2 the 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

12 Tandem MS - peptide fragmentation
low energy collision fragments the peptide cleavage usually occurs at the amide bond i.e. between residues 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 During 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 series However, 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

13 peptide fragmentation
How does amino acid sequencing on a mass spec work? Again the msms procedure is used The 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 event This cleavage most frequently occurs at a peptide bond in the backbone of the parent peptide chain So from each group of parent peptides of a selected mass, a “nested” set of fragments are produced The mass difference between 2 adjacent fragments (daughter fragments) corresponds to the residue that differs between them So the sequence of peptide can be determined by working along the ion series spelling out each amino-acid in the chain This 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 time The resulting amino-acid sequence can then be used in a database search e.g. BLAST to try and identify the protein The spectrum here shows the daughter ions produced from one parent peptide in the nested set, the mass difference between 2 adjacent fragments (peptide ions/daughter fragments) corresponds to the residue that differs between the two fragments So 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 protein Even 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 search the series of fragments corresponds to the sequence of the peptide

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

15 Many peptides are fragmented during a 60 minute run
On-line LC-MSMS on the Q-ToF2 : peptides from a single protein Survey Mass Spectrum (MS) - intact peptides detected in a 1 second survey scan Fragment Mass spectrum (MSMS) fragments from one peptide MSMS MSMS 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 MS/MS instruments can select a single peptide ion out of a spectrum by MS1 This peptide ion is fragmented by collision with inert gas The mass of the fragment ions is measured by MS2 = ToF

16 MSMS ions search data peak list (pkl) 920.9598 241.0128 2
: MSMS ions search data peak list (pkl) Peptide mass : charge state : intensity fragment mass : intensity

17 Examples of open access search tools
MASCOT 3 types of searches Aldente PMF search tool Phenyx an MS data analysis platform identification and characterization of proteins & peptides from mass spectrometry data

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

19 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

20 Cut-off score for significance is different for every search
Decoy database search Cut-off score for significance is different for every search

21 Number of matching peptides Protein score Protein name
Peptide score Expect value Protein Hit List The 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 value Experimental m/z transformed to a relative molecular mass Relative molecular mass calculated from the matched peptide sequence Difference (error) between the experimental and calculated masses Number of missed cleavage sites Ions 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 hit Sequence 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 peptide Different species

22 Predicted mass and predicted pI
Sequence coverage Only the peptide masses and their fragment ion masses are matched – the peptides themselves have not actually been sequenced

23 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

24 Download the MSMS files 1 to 4 onto the desktop
Click on the MS/MS Ions Search tool page

25 Standard search input your name & your e-mail use standard defaults
swissprot trypsin, 1 missed cleavage variable on Carbamidomethyl C variable on Oxidation M peptide charge +2, +3, +4 Copy MSMS files to desktop Browse to add file to search page Micromass PKL ESI-QUAD-TOF

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

27 Selected 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, Pages David E. Garfin Trends in Analytical Chemistry Volume 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 SPECTROMETRY Matthias Mann, Ronald C. Hendrickson, and Akhilesh Pandey Annual Review of Biochemistry July 2001, Vol. 70, pp Challenges 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 al Nature Jan 10;415(6868):141-7.

28 Development 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. Minden State-of-the-art in phosphoproteomics Proteomics 2005 Early View i.e. find it on the journals early view section of the web site Joerg Reinders, Albert Sickmann Global 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.

29 links 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 seminars proteome 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 Mass Spectrometry and Biotechnology Resource – lots of useful info – tutorials on de novo sequencing etc

30 Example of good quality peptide match

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

32 Example of poor quality peptide

33 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


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