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Biomedical subjects

J S Cottrell

Publications and source records attributed to J S Cottrell.

16 recordsLinked to original sources
PubMed2001

Interrogating the human genome using uninterpreted mass spectrometry data.

The public availability of a draft assembly of the human genome has enabled us to demonstrate, for the first time, the feasibility of searching a complete, unmasked eukaryotic genome using uninterpreted mass spectrometry data. A complex LC-MS/MS data set, containing peptides from at least 22 human proteins, was searched against a comprehensive, nonidentical protein database, an expressed sequence tag (EST) database, and the International Human Genome Project draft assembly of the human genome. The results from the three searches are compared in detail, and the merits of the different databases for this application are discussed. In the case of the EST database, the UniGene index provided a method of simplifying and summarising the search results. In the case of the genomic DNA, the presence of introns prevented matching of roughly one quarter of the spectra, but the technique can provide primary experimental verification of predicted coding sequences, and has the potential to identify novel coding sequences.

Algorithms↗
PubMed2001

Matching peptide mass spectra to EST and genomic DNA databases.

The use of mass spectrometry data to search molecular sequence databases is a well-established method for protein identification. The technique can be extended to searching raw genomic sequences, providing experimental confirmation or correction of predicted coding sequences, and has the potential to identify novel genes and elucidate splicing patterns.

Amino Acid Sequence↗
PubMed1999

Probability-based protein identification by searching sequence databases using mass spectrometry data.

Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be compared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.

Amino Acid Sequence↗
PubMed1995

Identification of myocardial proteins from two-dimensional gels by peptide mass fingerprinting.

Two-dimensional gels offer a powerful method for separating complex protein mixtures, but subsequent methods for analysing individual components, such as protein sequencing and Western immunoblotting, are laborious and slow. The identification of proteins can be accelerated by using a combination of protease digestion and matrix assisted laser desorption-mass spectrometry (MALDI-MS). The peptide mass spectrum of a protein represents a unique fingerprint determined by the amino acid sequence and the cleavage properties of the protease. Software has been developed so that peptide masses can be used to search a mass-based peptide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identification of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two-dimensional electrophoresis (2-DE) gels and analysed blind. Conditions for optimal trypsin digestion of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes are described. Mass data were generated from both Coomassie Brilliant Blue and sulforhodamine B-stained proteins, though the former required destaining prior to digestion. Alkylation of cysteine and oxidation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented to illustrate the advantages and disadvantages of this approach.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1994

Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry.

A rapid and sensitive method for sequencing oligosaccharides has been developed, using matrix-assisted laser desorption mass spectrometry to monitor the digestion of glycopeptides by specific exoglycosidases. Recombinant human tissue inhibitor of metalloproteinases (TIMP), which has two glycosylation sites, has been characterized to illustrate this new approach for obtaining site-specific information. Glycopeptides which span residues Asn30 and Asn78 were generated by tryptic digestion of 1 nmol of TIMP and separated by reverse-phase high-performance liquid chromatography. The oligosaccharide composition of the glycoforms was inferred from the observed mass shifts following digestion by peptide-N-glycosidase F. Composition and sequence were then elucidated by digestion with specific exoglycosidases, using a total of 200 pmol of each glycopeptide. Glycopeptides from well-characterized proteins, fetuin, alpha 1-acid glycoprotein, and tissue plasminogen activator were also analyzed to confirm exoglycosidase specificity for glycopeptides and establish the quantitative significance of the relative intensities of peaks in the mass spectra. Both TIMP glycosylation sites exhibited extensive heterogeneity comprising mainly fucosylated complex oligosaccharides, but in different proportions. The Asn78 site also contained 4.4% nonfucosylated mannose (Man4) oligosaccharide. The merits and limitations of this approach as a universal method for oligosaccharide analysis are discussed.

Amidohydrolases↗
PubMed1992

Sample immobilization protocols for matrix-assisted laser-desorption mass spectrometry.

Methods are described for the purification of proteins prior to analysis by matrix-assisted laser-desorption mass spectrometry. Contaminated protein samples were immobilized onto the surfaces of sample targets and rinsed. In general, a layer of electrosprayed nitrocellulose gave better results than the roughened gold surface of an untreated target. Using this approach, spectra could be obtained from low picomole quantities of protein in the presence of contaminants which did not inhibit the binding of the protein to the substrate.

Animals↗
PubMed1991

The analysis of underivatized oligosaccharides by matrix-assisted laser desorption mass spectrometry.

Matrix Assisted Laser Desorption Mass Spectrometry is shown to provide a rapid and sensitive technique for the analysis of underivatized oligosaccharides. Typical sample loading is 1 pmol and analysis time is around 5 minutes. Through the use of an internal standard, mass measurements are generally accurate to within 0.5 Da. The technique is particularly useful for the analysis of oligosaccharide mixtures released from glycoproteins.

Carbohydrate Conformation↗
PubMed1987

Improved detection limits in an organic mass spectrometer using a combination of matrix free FAB and photodiode array detection.

Scanning mass spectrometers suffer from the disadvantage of monitoring only one mass at any particular time in contrast to mass spectrographs which allow the simultaneous detection of an extended mass range. Historically such mass spectra were recorded by directing the ion beam onto a photographic plate which was subsequently developed and interpreted. The introduction of electronic devices such as the photodiode array enabled the development of imaging detectors capable of real time readout, so that a range of masses can be detected and rapidly processed using a computer system. This work uses such a detector in combination with a double focusing MS50RF mass spectrometer for the parallel detection of a limited mass range.

Mass Spectrometry↗
PubMed1987

Continuous-flow fast atom bombardment mass spectrometry.

The continuous-flow fast atom bombardment probe performs equally well with or without a high-performance liquid chromatography column producing clean spectra containing little or no background noise. Its function as a liquid chromatography-mass spectrometry interface for labile and involatile samples has been illustrated with reference to dansylated amino acids. The versatility of the new probe has been exemplified by on-line enzymatic peptide sequencing.

Amino Acids↗
PubMedDate not supplied

The analysis of myocardial proteins by infrared and ultraviolet laser desorption mass spectrometry.

The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.

Electrophoresis, Gel, Two-Dimensional↗
PubMedDate not supplied

Protein identification by peptide mass fingerprinting.

A mass spectrum of the peptide mixture resulting from the digestion of a protein by an enzyme can provide a fingerprint of great specificity--so specific, in fact, that it is often possible to identify the protein from this information alone, without ambiguity. The general approach is to take a small sample of the protein of interest and digest it with a proteolytic enzyme, such as trypsin. The resulting digest mixture is analyzed by mass spectrometry, the ionization techniques of choice being matrix-assisted laser desorption or electrospray ionization. The experimental mass values are then compared with a database of peptide mass values, calculated by applying the enzyme cleavage rules to the entries in one of the major collections of sequence data, such as SwissProt or PIR. By using an appropriate scoring algorithm, the closest match or matches can be identified. If the "unknown" protein was present in the sequence database, then the goal is to identify that precise entry. If the sequence database does not contain the unknown protein, then a successful search will identify those entries that exhibit the closest sequence homology, often equivalent proteins from related species. An inverted strategy, in which experimental peptide mass fingerprints are accumulated in a database, has significant potential for identifying coding regions within sequence data from genomic DNA and, in doing so, correlating the genes with their expressed proteins. This review aims to provide an overview of the technique, compare the different database matching algorithms which have been described in the literature, and discuss the practical and theoretical factors which influence identification accuracy.

Algorithms↗