From usenet.ucs.indiana.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!europa.eng.gtefsd.com!uunet!pipex!sunic!corax.udac.uu.se!zeta.bmc.uu.se!daresbury!news!ajb Thu Aug 26 19:18:39 EST 1993 Article: 3120 of bionet.software Path: usenet.ucs.indiana.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!europa.eng.gtefsd.com!uunet!pipex!sunic!corax.udac.uu.se!zeta.bmc.uu.se!daresbury!news!ajb From: ajb@s-crim1.dl.ac.uk (Alan Bleasby) Newsgroups: bionet.software Subject: [ANNOUNCE] MOWSE database email server Date: 26 Aug 1993 23:46:52 GMT Organization: SERC Daresbury Lab, Warrington, U.K. Lines: 665 Distribution: bionet Message-ID: NNTP-Posting-Host: s-crim1.dl.ac.uk A peptide mass fingerprint email server service is now available by emailing mowse@dl.ac.uk The help file, available from this address, is reproduced below and describes the database and how to access it. It allows identification of known proteins from a set of molecular weights (mass spec) determined after proteolytic digests. Alan Bleasby SERC Daresbury Laboratory ******************************** The MOWSE peptide mass database: ******************************** Imperial Cancer Research Fund and SERC Daresbury Laboratory D.J.C. Pappin, P. Hojrup and A.J. Bleasby 'Rapid Identification of Proteins by Peptide-Mass Fingerprinting'. Current Biology (1993), vol 3, 327-332. InterNet server version: Table of Contents: [1] Background. [2] Construction of the MOWSE database. [2.1] Source database. [2.2] Calculation of Molecular weight fragments. [3] Running database searches via e_mail. [4] Example of mail query format. [5] Results listing. [6] Database structure. [6.1] MOWSE database structure. [6.2] The MW primary fragment molecular weight file. [6.3] The MDX file OWL entry index. [6.4] The SMW whole sequence molecular weight file. [6.5] Program Requirements. [6.6] MOWSE Scoring scheme. [6.7] Simulation studies. [7] General references. [1] Background: Determination of molecular weight has always been an important aspect of the characterization of biological molecules. Protein molecular weight data, historically obtained by SDS gel electrophoresis or gel permeation chromatography, has been used establish purity, detect post-translational modification (such as phosphorylation or glycosylation) and aid identification. Until just over a decade ago, mass spectrometric techniques were typically limited to relatively small biomolecules, as proteins and nucleic acids were too large and fragile to withstand the harsh physical processes required to induce ionization. This began to change with the development of 'soft' ionization methods such as fast atom bombardment (FAB)[1], electrospray ionisation (ESI) [2,3] and matrix-assisted laser desorption ionisation (MALDI)[4], which can effect the efficient transition of large macromolecules from solution or solid crystalline state into intact, naked molecular ions in the gas phase. As an added bonus to the protein chemist, sample handling requirements are minimal and the amounts required for MS analysis are in the same range, or less, than existing analytical methods. As well as providing accurate mass information for intact proteins, such techniques have been routinely used to produce accurate peptide molecular weight 'fingerprint' maps following digestion of known proteins with specific proteases. Such maps have been used to confirm protein sequences (allowing the detection of errors of translation, mutation or insertion), characterise post-translational modifications or processing events and assign disulphide bonds [5,6]. Less well appreciated, however, is the extent to which such peptide mass information can provide a 'fingerprint' signature sufficiently discriminating to allow for the unique and rapid identification of unknown sample proteins, independent of other analytical methods such as protein sequence analysis. The following text describes the construction and use of the MOWSE peptide mass database (for MOlecular Weight SEarch) at the SERC Daresbury Laboratory. Practical experience has shown that sample proteins can be uniquely identified using as few as 3- 4 experimentally determined peptide masses when screened against a fragment database derived from over 50,000 proteins. Experimental errors of a few Daltons are tolerated by the scoring algorithms, permitting the use of inexpensive time-of-flight mass spectrometers. As with other types of physical data, such as amino acid composition or linear sequence, peptide masses can clearly provide a set of determinants sufficiently unique to identify or match unknown sample proteins. Peptide mass fingerprints can prove as discriminating as linear peptide sequence, but can be obtained in a fraction of the time using less material. In many cases, this allows for a rapid identification of a sample protein before committing to protein sequence analysis. Fragment masses also provide structural information, at the protein level, fully complementary to large-scale DNA sequencing or mapping projects [7,8,9]. [2] Construction of the MOWSE database. [2.1] Source database. MOWSE was created from the OWL non-redundant composite protein sequence database [10,11]. The first InterNet release (version 20.1) contains some 61,000 protein entries, generating approximately 15,000,000 peptide fragments. The MOWSE fragment database will be updated with each new release of the parent OWL database (every 2 months or so). [2.2] Calculation of Molecular weight fragments. For each entry in the source OWL database, MOWSE derives both whole sequence molecular weight and calculated peptide molecular weights for complete digests using the range of cleavage reagents and rules detailed in Table 1. Cleavage is disallowed if the target residue is followed by proline (except for CNBr or Asp N). Glu C (S. aureus V8 protease) cleavages are also inhibited if the adjacent residue is glutamic acid. Peptide mass calculations are based entirely on the linear sequence and use the average isotopic masses of amide-bonded amino acid residues (IUPAC 1987 relative atomic masses). To allow for N-terminal hydrogen and C-terminal hydroxyl the final calculated molecular weight of a peptide of N residues is given by the equation: N __ \ / Residue mass + 18.0153 -- n=1 Molecular weights are rounded to the nearest integer value before being entered into the database. Cysteine residues are calculated as the free thiol, anticipating that samples are reduced prior to mass analysis. CNBr fragments are calculated as the homoserine lactone form. Information relating to post- translational modification (phosphorylation, glycosylation etc.) is not incorporated into calculation of peptide masses. Reagent no. Reagent Cleavage rule 1 Trypsin C-term to K/R 2 Lys-C C-term to K 3 Arg-C C-term to R 4 Asp-N N-term to D 5 V8-bicarb C-term to E 6 V8-phosph C-term to E/D 7 Chymotrypsin C-term to F/W/Y/L/M 8 CNBr C-term to M Table 1: Cleavage reagents modelled by MOWSE. [3] Running database searches by e_mail: ******************************************************************** Search queries should be mailed to mowse@daresbury.ac.uk (short form mowse@dl.ac.uk). Search results will be returned directly to your e_mail address. Comments, please, to mbdpn@s-crim1.dl.ac.uk. ******************************************************************** The 'subject' field of your email message is irrelevant - all parameters must be specified in the body of the message. The relevant syntax is given below. Some lines are compulsory, others are optional (see the description of parameters section). All text is case-insensitive, and MOWSE expects integer data. Non-exponential floating point syntax is acceptable, but MOWSE will round the data to the nearest integer. Whitespace is ignored in an intuitive way. MOWSE recognises the following command lines which are further described below Begin Reagent Tolerance SeqMW Filter Datastart Dataend The order of lines is irrelevant with the exception of 'begin' and the 'datastart/dataend' commands (see below). If multiple instances of a command occur then only the FIRST instance will be recognised Begin Every search query MUST start with a 'begin' line. There should only be one 'begin' line and all other commands & data should immediately follow. Reagent Every search query MUST specify a 'reagent' line. The word 'reagent' must be followed by one of the supported cleavage reagents. These are: Trypsin Lys-C Arg-C Asp-N V8-bicarb V8-phosph Chymotrypsin CNBr A typical reagent line is therefore of the form: reagent trypsin Tolerance This line is optional. The supplied number specifies the error allowed for mass accuracy of experimental mass determination. If no figure is specified, a default tolerance of 2 Daltons will be assumed. If you wish to specify a different tolerance then follow the word 'tolerance' with the required number of Daltons e.g. tolerance 1 In this case, supplied peptide masses will be matched to +/- 1 Daltons. Values of 2-4 are suggested for data obtained by laser- desorption TOF instruments. Accuracies of +/- 2 Daltons or better are generally only possible using an appropriate internal standard (e.g. oxidised insulin B chain) with TOF instruments. For electrospray or FAB data, a value of 1 can be selected in most cases. If you have real confidence in mass determination, specify '0' (zero) to limit matches to the nearest integer value (effectively +/- 0.5 Daltons). Discrimination is significantly improved by the selection of a small error tolerance. SeqMW This optional line allows you to give the molwt of the whole protein (if known). This allows you to limit the search to proteins of this molwt plus/minus a 'limit' (see below). If unspecified, a whole protein molwt of 0 is assumed which MOWSE interprets as "search the whole database". This will include all proteins up to the maximum size of just under 700,000 Daltons. You can specify any molwt in Daltons with this command e.g. SeqMW 90000 Filter This optional line is used in conjunction with the SeqMW command and is meaningless without it. It specifies a percentage. Only proteins of the given SeqMW +/- this percentage will be searched. If a SeqMW is specified but Filter is unspecified then Filter will default to 25%. To specify a percentage of 30% use: Filter 30 In this case, a molecular weight of 90,000 Daltons was specified and the selection of 30 for the filter restricts the search to those proteins with masses from 63,000 to 117,000 Daltons. A value of 25 is suggested for initial searches, which can be progressively widened for subsequent search attempts if no matches are found. Discrimination is best when the filter percentage is narrow, but some Mw estimates (particularly from SDS gels) should be given considerable allowance for error. Help If this line appears anywhere in the text body then only this document will be emailed to you! Specifying molecular weight data: Datastart This line is compulsory and must mark the beginning of the fragment molecular weight data. The molecular weights must follow this line, each molecular weight being on a line of its own. Masses (M not M[H+]) are accepted in any order (ascending,descending or mixed). Peptide masses can be entered as integers or floating-point values, the latter being rounded to the nearest integer value for the search. It is suggested that peptide masses should be selected from the range 700-4000 Daltons. This range balances the fact that very small peptides give little discrimination and minimizes the frequency of partially-cleaved peptides. As a general rule, users are advised to identify and remove peptide masses resulting from autodigestion of the cleavage enzyme (e.g tryptic fragments of trypsin), best obtained by MS analysis of control digests containing only the enzyme. Dataend This line marks the end of the fragment data and must be on a line of its own. If the data block is at the end of the mail query then this line is optional. [4] Example mail query: The following example text should form the body of a typical e_mail query: Begin Reagent Trypsin Tolerance 2 SeqMW 90000 Filter 30 Datastart 813 845 880 940 1055 1178 1380 1520 1562 1648 1777 2079 Dataend ********************************************************************* NOTE: The described structure will allow for multiple search requests per e_mail message. This feature is NOT presently supported and users are asked to submit separate e_mail messages for each search request. ********************************************************************* [5] Results listing (InterNet version). The MOWSE search program outputs a listing file containing the following information. Specified search parameters. Includes all specified parameters such as digest reagent, specified error tolerance, specified intact protein Mw and Mw filter percentage. All supplied peptide Mws are listed in descending order, followed by the total number of entries scanned during the search. Short 'hit' listing. The top 30 scoring proteins are then listed in descending order, details including the OWL entry code and brief text identifiers. Details are limited to the top 30 scores as a deliberate compromise to keep the result listings as short as possible for e_mail return. Detailed 'hit' listing. The top 30 entries are then listed in more detail.The first line includes the OWL entry code, the MOWSE search score (typically a few powers of 10), the 'hit' protein Mw and finally an 'accuracy' ratio calculated by dividing 'hits' by the total number of peptides used for the search. This cannot be used to ascribe significance, but experience has shown that anything below 0.3 is generally not worth pursuing. Line 2 is the OWL text identifier. Subsequent lines list 'hit' and 'miss' peptides, with the appropriate start, end and corresponding sequences of correct peptide matches. One problem for InterNet users is that the OWL sequence database is only available to UK users. We have thus tried to ensure that the 'hit' listing contains sufficient information, either in the text description or matched peptide sequences to allow users to identify target proteins within their own databases. If this proves a major problem, then we will include other text information (EMBL accession numbers, for example) to assist identification. [6] Database structure. [6.1] MOWSE database structure. The database consists of three binary files: i) MOWSE.MW The primary file containing the fragment molecular weights. ii) MOWSE.MDX Index file relating OWL identifier codes to the molecular weight information in the primary Mw file. iii) MOWSE.SMW Calculated molecular weights of intact OWL sequences. The query program accesses the binary information transparently from the user viewpoint. In the internal representation the molecular weight (and other) integers are stored as 4-byte machine specific quantities. The binary files can be transferred between machines of the same 'endian' nature, but 'cross-endian' transfer is not possible. The MOWSE software allows recreation of the files on any platform supporting a standard C language compiler. The organisation of the database files is described below. [6.2] The MW primary fragment molecular weight file. Fragment molecular weight entries in this file map sequentially to the order of entries within the source (OWL) protein sequence file. Each MW file entry consists of 4 blocks and are shown below. The MW entries are catenated. Block 1 OWL Entry Code 20 bytes Block 2 OWL Title Line 80 bytes Block 3 Reagent Table 80 bytes Block 4 Reagent 1 4 byte Reagent 2 Reagent 3 - - - Reagent 8 The OWL entry code is the unique identifier of the source protein sequence within the OWL database. The code is padded to 20 bytes using null characters. The title line contains the descriptive text of the source protein sequence as given in OWL (null terminated). When the OWL title line is longer than 80 characters, the MW entry is truncated. Block 3 is a table of 20 consecutive 4-byte integers, one for each allowed reagent in the current MOWSE implementation. The order of the integers follows the order given in Table 1. Each integer holds the number of fragments derived from the target sequence using the associated enzyme (e.g. the fourth integer represents the number of fragments produced by the theoretical complete digest of a sequence with Arg-C). Unused slots (9-20) are assigned zero value. The final block consists of 4-byte integers holding the fragment molecular weights. Again, the lists mirror the order of the enzymes given in Table 1. Fragment peptides for each enzyme are sorted on the basis of continually decreasing calculated molecular weight. [6.3] The MDX file OWL entry index. The MDX file consists of three blocks as shown below: Block 1 Header Information Block 2 FTELL entries Block 3 Buckets HEADER: This contains six 4-byte binary integers containing a) The number of OWL database entries b) The maximum length of a unique identifier code c) The number of buckets (see text) d) The start position of Block 2 within the MDX file. e) Reserved f) The start position of Block 3 within the MDX file FTELL ENTRIES: This is a block of 4-byte binary integers giving the start position of MW file entries. The list reflects the sequential order of the entries in the MW file. The positions are the displacement, in bytes, of the start of an entry from the beginning of the MW file. BUCKETS: Given a unique OWL identifier, a hashing algorithm is used to quickly locate fragment molecular weights. The initial twelve characters of the identifier are grouped into consecutive binary pairs thereby yielding six 2-byte integers. These are hashed using the equation: T = (int4 xor int2) * 26 + (int1 xor int3) * 23 + (int0 xor int5) Identifiers having less than twelve characters are padded out with spaces. The final hash value 'B' is given by: B = T modulo PRIME where the value B represents a 'bucket' into which the identifier will slot. The number 'PRIME' is a prime number giving the number of buckets within the file and is the third integer in the header block. Each bucket is 512 bytes long and contains a maximum of thirty two 16-bit entries. Each bucket entry consists of the twelve bytes of unique OWL identifier code (truncated or padded with spaces) followed by a 4-byte integer. The integer is the sequential position of the MOWSE entry (the first entry is represented by the value 1). The MDX indexing software chooses the prime number value such that there is no bucket containing more than the allowed number of entries. In order to locate the start position of an entry in the MW file the entry code is hashed to determine the bucket number. The relevant bucket is searched to find the sequential position of the entry. The sequential position is then used as an index into Block 2 to find the displacement of the MW entry with respect to the start of the file. [6.4] The SMW whole sequence molecular weight file. Each sequence molecular weight is represented as a 4-byte integer. The molecular weights are stored in the same order as the entries for each protein in the primary fragment MW file. [6.5] Program Requirements. The MOWSE search program accepts a single text file containing a list of experimentally-determined masses, generally selected from the range 700-4,000 Daltons to reduce the influence of partial cleavage products. The program outputs a ranked hit list comprising the top 30 scores, with information including the OWL entry name, text identifiers, final accumulated scores, matching peptide sequences and hit versus miss tallies. User-selectable search parameters include an error tolerance (default 1 2 Daltons), selection of the enzyme or reagent used and an intact protein Mw (optional, if known). For each peptide Mw entry in the data file, MOWSE matches individual fragment molecular weights (FMWs) with database entry molecular weights (DBMWs). A 'hit' is scored when the following criterion is met: DBMW-tolerance-1 < FMW < DBMW+tolerance+1 If an intact protein Mw is specified (SMW) then the program prompts for a molecular weight filter percentage (MWFP). MOWSE then restricts the search to those entries which match the following criteria: R = SMW x MWFP / 100 0 < SMW-R < MOWSE entry Mol.wt. < SMW+R Default search parameters are a tolerance of +/- 2 Daltons, intact Mw specified and the MWFP set to 25. [6.6] MOWSE Scoring scheme. The final scoring scheme is based on the frequency of a fragment molecular weight being found in a protein of a given range of molecular weight. OWL database sequence entries were initially grouped into 10 kDalton intact molecular weight intervals. For each 10 kDalton protein interval, peptide fragment molecular weights were assigned to cells of 100 Dalton intervals. The cells therefore contained the number of times a particular fragment molecular weight occurred in a protein of any given size. This operation was performed for each enzyme. Cell frequency values were calculated by dividing each cell value by the total number of peptides in each 10 kD protein interval. Cell frequency values for each 10 kDalton interval were then normalised to the largest cell value (Fmax), with all the cell values recalculated as: Cell value = Old value / Fmax to yield floating point numbers between 0 and 1. These distribution frequency values, calculated for each cleavage reagent, were then built into the MOWSE search program. For every database entry scanned, all matching fragments contribute to the final score. In the current implementation, non-matching fragments are ignored (neutral). For each matching peptide Mw a score is assigned by looking up the appropriate normalised distribution frequency value. In the case of multiple 'hits' in any one target protein (i.e. more than one matching peptide Mw), the distribution frequency scores are multiplied. The final product score is inverted and then normalised to an 'average' protein Mw of 50 kDaltons to reduce the influence of random score accumulation in large proteins (>200 kDaltons). The final score is thus calculated as: Score = 50/(Pn x H) Where Pn is the product of n distribution scores and H the 'hit' protein molecular weight in kD. Important consequences of this type of scoring scheme are that matches with peptides of higher Mw carry more scoring weight, and that the non-random distribution of fragment molecular weights in proteins of different sizes is compensated for. [6.7] Simulation studies. In a simulation of scoring properties, 100 test proteins with masses between 10 kD and 100 kD were randomly selected from the OWL sequence database. The sets of all possible tryptic peptide masses for each protein were randomized and database searches performed with increasing numbers of fragments (default search parameters) until the test protein reached the top of the ranked scoring list. 99% of the test proteins were correctly identified using only five peptides or less (mean=3.6 peptides), with one example requiring six. These figures were surprisingly small considering that some of the proteins in the test sample generated more than 100 possible tryptic fragments. All 100 test examples were identified using 30% or less of the maximum number of available peptides. This distribution was somewhat dependent on protein size, as smaller proteins generally yield fewer peptide fragments. Thus, all proteins of 30 kD and over were identified using 13% or less of possible fragments (1 in 8), with all proteins of 40 kD and above requiring less than 10% (1 in 10). In this latter group, therefore, more than 90% of the potential information (all possible peptides) was redundant. For the simulation a 'unique' identification required matching not only of protein type (e.g. globin) but correct discrimination of type, species, and isoform or isozyme. Discrimination could be further improved by reducing the error tolerance to only +/- 1 Dalton (mean=2.7 peptides). Such accuracies are easily bettered using more sophisticated ESI/quadrupole or high-field FAB spectrometers, but the default search value (+/- 2 Daltons) compensates for the reduced accuracy obtainable from the smaller time-of-flight (TOF) instruments. Mass accuracies better than +/- 1 Dalton were not essential, and in fact the error tolerance could be relaxed to +/- 5 Daltons in many cases with little degradation in performance. The simulation thus clearly demonstrated the high degree of discrimination afforded by relatively few peptide masses, even with generous allowance for error. [7] General references. 1: Barber M, Bordoli RS, Sedgwick RD, Tyler AN: Fast atom bombardment of solids: a new ion source for mass spectrometry. J Chem Soc Chem Commun 1981, 7: 325-327. 2: Dole M, Mack LL, Hines RL, Mobley RC, Ferguson LD, Alice MB: Molecular beams of macroions. J Chem Phys 1968, 49:2240-2249. 3: Meng CK, Mann M, Fenn JB: Of protons or proteins. Z Phys D 1988, 10: 361-368. 4: Karas M, Hillenkamp F: Laser desorption ionisation of proteins with molecular masses exceeding 10,000 Daltons. Analytical Chemistry 1988, 60:2299-2301. 5: Morris H, Panico M, Taylor GW: FAB-mapping of recombinant-DNA protein products. Biochem Biophys Res Commun 1983, 117:299-305. 6: Morris H, Greer FM: Mass spectrometry of natural and recombinant proteins and glycoproteins. Trends in Biotechnology 1988, 6:140-147. 7: Weissenbach J, Gyapay G, Dib C, Vignal J, Morissette J, Millasseau P, Vaysseix G, Lathrop M: A second generation linkage map of the human genome. Nature 1992, 359:794-801. 8: Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF, Kerlavage AR, McCombie WR, Venter JC: Complementary DNA sequencing: expressed sequence tags and human genome project. Science 1991, 252:1651-1656. 9: Lehrach H, Drmanac R, Hoheisel J, Larin Z, Lennon G, Monaco AP, Nizetic D, Zehetner G, Poustka A: Hybridization fingerprinting in genome mapping and sequencing. In Genome Analysis Volume 1: Genetic and Physical Mapping. Cold Spring Harbor Laboratory Press; 1990:39-81 . 10: Akrigg D, Bleasby AJ, Dix NIM, Findlay JBC, North ACT, Parry- Smith D, Wootton JC, Blundell TI, Gardner SP, Hayes F, Sternberg MJE, Thornton JM, Tickle IJ, Murray-Rust P: A protein sequence/structure database. Nature 1988, 335:745-746. 11: Bleasby AJ, Wootton JC: Construction of validated, non- redundant composite protein databases. Protein Engineering 1990, 3:153-159.