Proteomicists or individuals involved in proteomics (and related fields in academia and industry) need to immediately scan literature; access databases; use tools and software; find information on collections, educational resources, and research groups; and exploit that information for their scientific projects. In response to these needs, Proteomicist.org serve as a quick open-access, online guide to the proteomics knowledge. Intended to be a one-stop location with maximum coverage of the field as possible, this site performs various functions: delivering freshest content from leading proteomics sources; highlighting key contributions to the development of proteomics; providing a well-organised and curated proteomics web directory, direct references for proteomics-related terms, multiple site search, and more.
Reagents & Kits (4)
Mascot by Matrix Science
Mascot is a proprietary identification program available from Matrix Science. It is a powerful search engine that uses mass spectrometry data to identify proteins from primary sequence databases.
The mission and goals of Clinical Proteomics are to provide a scholarly forum for novel scientific research in the field of translational proteomics.
Proteomic Forum 2011
Proteomic Forum will bring together leading experts from all over the world and provide a critical mass in proteomics technologies, molecular and cellular proteomics as well as clinical proteomics.
Journal of Proteome Research (JPR)
JPR publishes content encompassing all aspects of global protein analysis and function, emphasizing the synergy between physical and life sciences resulting in a multidisciplinary approach to the understanding of biological processes.
SEQUEST is the original, and still one of the leading, programs that correlates uninterpreted tandem mass spectra of peptides against amino acid sequences from sequence databases.
X! Tandem is a open source software that can match tandem mass spectra with peptide sequences, in a process that has come to be known as protein identification.
ProteoIQ by BioInquire
ProteoIQ is a software package for the post-analysis of proteomic dataset results derived from Mascot, SEQUEST, or X!Tandem search results.
Current Proteomics is an essential journal for everyone involved in proteomics and related fields in both academia and industry.
X! Tandem is a open source software that can match tandem mass spectra with peptide sequences, in a process that has come to be known as protein identification.
Mascot by Matrix Science
Mascot is a proprietary identification program available from Matrix Science. It is a powerful search engine that uses mass spectrometry data to identify proteins from primary sequence databases.
Quansys Biosciences, a Spendlove Research company, is dedicated to the development of protein arrays to aide researchers and clinicians in better understanding, diagnosing, and treating disease.
Protea Biosciences, a leader in innovating protein sample preparation, is dedicated to developing new, cutting edge products to improve the quality of data obtained from valuable biological samples.
The 20 years of PROSITE.
Nucleic Acids Res. 2008 Jan;36(Database issue):D245-9
Authors: Hulo N, Bairoch A, Bulliard V, Cerutti L, Cuche BA, de Castro E, Lachaize C, Langendijk-Genevaux PS, Sigrist CJ
PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. In this article, we describe the implementation of a new method to assign a status to pattern matches, the new PROSITE web page and a new approach to improve the specificity and sensitivity of PROSITE methods. The latest version of PROSITE (release 20.19 of 11 September 2007) contains 1319 patterns, 745 profiles and 764 ProRules. Over the past 2 years, about 200 domains have been added, and now 53% of UniProtKB/Swiss-Prot entries (release 54.2 of 11 September 2007) have a PROSITE match. PROSITE is available on the web at: http://www.expasy.org/prosite/.
PMID: 18003654 [PubMed - indexed for MEDLINE]
The European Proteomics Association (EuPA) is in the field. Report of the formal inauguration of the European Proteome Association (Munich, Germany, August 29, 2005).
Proteomics. 2005 Dec;5(18):4648-50
Authors: Kleinhammer C, Corthals GL
After one year of preparation the European Proteomics Association (EuPA) was formally inaugurated on August 29, 2005, on the occasion of the 4(th) HUPO World Congress in Munich, Germany. Delegates from 16 European countries elected Dr. Friedrich Lottspeich, President of the German Proteome Society, as the first EuPA President. The EuPA Board also comprises Professor Mathias Uhlen as Vice President, along with Professor Michael Dunn (Ireland), Professor Concha Gil (Spain), Dr. Jean Charles Sanchez (Switzerland) and Professor Pier Giorgio Righetti (Italy) as Coordinators for the presently defined focus activities of the EuPA. The general objectives of the EuPA are to promote proteomic activities throughout Europe, emphasising the benefits and contribution of proteomics to biological researchers, industry, the general public and politicians.
PMID: 16281188 [PubMed - indexed for MEDLINE]
Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.
Proteomics. 2005 Nov;5(17):4327-37
Authors: Nilsson P, Paavilainen L, Larsson K, Odling J, Sundberg M, Andersson AC, Kampf C, Persson A, Al-Khalili Szigyarto C, Ottosson J, Björling E, Hober S, Wernérus H, Wester K, Pontén F, Uhlen M
A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.
PMID: 16237735 [PubMed - indexed for MEDLINE]
PRIDE: the proteomics identifications database.
Proteomics. 2005 Aug;5(13):3537-45
Authors: Martens L, Hermjakob H, Jones P, Adamski M, Taylor C, States D, Gevaert K, Vandekerckhove J, Apweiler R
The advent of high-throughput proteomics has enabled the identification of ever increasing numbers of proteins. Correspondingly, the number of publications centered on these protein identifications has increased dramatically. With the first results of the HUPO Plasma Proteome Project being analyzed and many other large-scale proteomics projects about to disseminate their data, this trend is not likely to flatten out any time soon. However, the publication mechanism of these identified proteins has lagged behind in technical terms. Often very long lists of identifications are either published directly with the article, resulting in both a voluminous and rather tedious read, or are included on the publisher's website as supplementary information. In either case, these lists are typically only provided as portable document format documents with a custom-made layout, making it practically impossible for computer programs to interpret them, let alone efficiently query them. Here we propose the proteomics identifications (PRIDE) database (http://www.ebi.ac.uk/pride) as a means to finally turn publicly available data into publicly accessible data. PRIDE offers a web-based query interface, a user-friendly data upload facility, and a documented application programming interface for direct computational access. The complete PRIDE database, source code, data, and support tools are freely available for web access or download and local installation.
PMID: 16041671 [PubMed - indexed for MEDLINE]
Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.
Mol Cell Proteomics. 2004 Dec;3(12):1154-69
Authors: Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ
We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.
PMID: 15385600 [PubMed - indexed for MEDLINE]
SWISS-2DPAGE, ten years later.
Proteomics. 2004 Aug;4(8):2352-6
Authors: Hoogland C, Mostaguir K, Sanchez JC, Hochstrasser DF, Appel RD
The SWISS-2DPAGE database was established in 1993 and is maintained collaboratively by the Swiss Institute of Bioinformatics (SIB) and the Biomedical Proteomics Research Group (BPRG) of the Geneva University Hospital. During these years, SWISS-2DPAGE underwent constant modification and improvement. Current content includes about 4000 identified spots corresponding to 1200 different protein entries in 36 reference maps from human, mouse, Arabidopsis thaliana, Dictyostelium discoideum, Escherichia coli, Saccharomyces cerevisiae and Staphylococcus aureus origins. With a high level of annotation and integration with other relevant databases, SWISS-2DPAGE is a reference source in the proteomics world. Queries to SWISS-2DPAGE database currently reach 1000 hits per day.
PMID: 15274128 [PubMed - indexed for MEDLINE]
A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
Anal Chem. 2004 Jul 15;76(14):4193-201
Authors: Liu H, Sadygov RG, Yates JR
Proteomic analysis of complex protein mixtures using proteolytic digestion and liquid chromatography in combination with tandem mass spectrometry is a standard approach in biological studies. Data-dependent acquisition is used to automatically acquire tandem mass spectra of peptides eluting into the mass spectrometer. In more complicated mixtures, for example, whole cell lysates, data-dependent acquisition incompletely samples among the peptide ions present rather than acquiring tandem mass spectra for all ions available. We analyzed the sampling process and developed a statistical model to accurately predict the level of sampling expected for mixtures of a specific complexity. The model also predicts how many analyses are required for saturated sampling of a complex protein mixture. For a yeast-soluble cell lysate 10 analyses are required to reach a 95% saturation level on protein identifications based on our model. The statistical model also suggests a relationship between the level of sampling observed for a protein and the relative abundance of the protein in the mixture. We demonstrate a linear dynamic range over 2 orders of magnitude by using the number of spectra (spectral sampling) acquired for each protein.
PMID: 15253663 [PubMed - indexed for MEDLINE]
The proteomics standards initiative.
Proteomics. 2003 Jul;3(7):1374-6
Authors: Orchard S, Hermjakob H, Apweiler R
The Proteomics Standards Initiative (PSI) aims to define community standards for data representation in proteomics and to facilitate data comparison, exchange and verification. Progress has been made in the development of common standards for data exchange in the fields of both mass spectrometry and protein-protein interaction. A proteomics-specific extension is being created for the emerging American Society for Tests and Measurements mass spectrometry standard, which will be supported by manufacturers of both hardware and software. A data model for proteomics experimentation is under development and discussions on a public repository for published proteomics data are underway. The Protein-Protein Interactions group expects to publish the Level 1 PSI data exchange format for protein-protein interactions soon and discussions as to the content of Level 2 have been initiated.
PMID: 12872238 [PubMed - indexed for MEDLINE]
HUPO (Human Proteome Organization) 1st World Congress. 21-24 November 2002, Versailles, France. Abstracts.
Mol Cell Proteomics. 2002 Sep;1(9):651-752
PMID: 12474872 [PubMed - indexed for MEDLINE]
Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
Mol Cell Proteomics. 2002 May;1(5):376-86
Authors: Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M
Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
PMID: 12118079 [PubMed - indexed for MEDLINE]
Creating the gene ontology resource: design and implementation.
Genome Res. 2001 Aug;11(8):1425-33
The exponential growth in the volume of accessible biological information has generated a confusion of voices surrounding the annotation of molecular information about genes and their products. The Gene Ontology (GO) project seeks to provide a set of structured vocabularies for specific biological domains that can be used to describe gene products in any organism. This work includes building three extensive ontologies to describe molecular function, biological process, and cellular component, and providing a community database resource that supports the use of these ontologies. The GO Consortium was initiated by scientists associated with three model organism databases: SGD, the Saccharomyces Genome database; FlyBase, the Drosophila genome database; and MGD/GXD, the Mouse Genome Informatics databases. Additional model organism database groups are joining the project. Each of these model organism information systems is annotating genes and gene products using GO vocabulary terms and incorporating these annotations into their respective model organism databases. Each database contributes its annotation files to a shared GO data resource accessible to the public at http://www.geneontology.org/. The GO site can be used by the community both to recover the GO vocabularies and to access the annotated gene product data sets from the model organism databases. The GO Consortium supports the development of the GO database resource and provides tools enabling curators and researchers to query and manipulate the vocabularies. We believe that the shared development of this molecular annotation resource will contribute to the unification of biological information.
PMID: 11483584 [PubMed - indexed for MEDLINE]
Human genomes, public and private.
Nature. 2001 Feb 15;409(6822):745
PMID: 11236960 [PubMed - indexed for MEDLINE]
The human genome. Science genome map.
Science. 2001 Feb 16;291(5507):1218
PMID: 11233443 [PubMed - indexed for MEDLINE]
Printing proteins as microarrays for high-throughput function determination.
Science. 2000 Sep 8;289(5485):1760-3
Authors: MacBeath G, Schreiber SL
Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.
PMID: 10976071 [PubMed - indexed for MEDLINE]
Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.
Nat Biotechnol. 1999 Oct;17(10):994-9
Authors: Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R
We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.
PMID: 10504701 [PubMed - indexed for MEDLINE]
Direct analysis of protein complexes using mass spectrometry.
Nat Biotechnol. 1999 Jul;17(7):676-82
Authors: Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JR
We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.
PMID: 10404161 [PubMed - indexed for MEDLINE]
Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.
Anal Chem. 1999 Jun 1;71(11):2076-84
Authors: Jensen PK, Pasa-Toli? L, Anderson GA, Horner JA, Lipton MS, Bruce JE, Smith RD
Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress etc.), or disease state(s). Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes and inference of protein function. Thus, much faster, more sensitive, and precise capabilities for the characterization of cellular constituents are desired. We describe progress in the development and initial application of the powerful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the proteome of the model system Escherichia coli. Isotope depletion of the growth media has been used to improve mass measurement accuracy, and the comparison of CIEF-FTICR results for the analysis of cell lysates harvested from E. coli cultured in normal and isotopically depleted media are presented. The initial studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of approximately 300 ng of E. coli proteins in a single CIEF-FTICR analysis.
PMID: 10366890 [PubMed - indexed for MEDLINE]
Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.
Electrophoresis. 1997 Oct;18(11):2071-7
Authors: Unlü M, Morgan ME, Minden JS
We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.
PMID: 9420172 [PubMed - indexed for MEDLINE]
Expression monitoring by hybridization to high-density oligonucleotide arrays.
Nat Biotechnol. 1996 Dec;14(13):1675-80
Authors: Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H, Brown EL
The human genome encodes approximately 100,000 different genes, and at least partial sequence information for nearly all will be available soon. Sequence information alone, however, is insufficient for a full understanding of gene function, expression, regulation, and splice-site variation. Because cellular processes are governed by the repertoire of expressed genes, and the levels and timing of expression, it is important to have experimental tools for the direct monitoring of large numbers of mRNAs in parallel. We have developed an approach that is based on hybridization to small, high-density arrays containing tens of thousands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide chemistry. RNAs present at a frequency of 1:300,000 are unambiguously detected, and detection is quantitative over more than three orders of magnitude. This approach provides a way to use directly the growing body of sequence information for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize small arrays containing hundreds of thousands of specifically chosen oligonucleotides, the method is readily scalable to the simultaneous monitoring of tens of thousands of genes.
PMID: 9634850 [PubMed - indexed for MEDLINE]
Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
Anal Chem. 1996 Mar 1;68(5):850-8
Authors: Shevchenko A, Wilm M, Vorm O, Mann M
Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
PMID: 8779443 [PubMed - indexed for MEDLINE]
Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.
Nature. 1996 Feb 1;379(6564):466-9
Authors: Wilm M, Shevchenko A, Houthaeve T, Breit S, Schweigerer L, Fotsis T, Mann M
Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.
PMID: 8559255 [PubMed - indexed for MEDLINE]
Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it.
Biotechnol Genet Eng Rev. 1996;13:19-50
Authors: Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, Williams KL
PMID: 8948108 [PubMed - indexed for MEDLINE]
Error-tolerant identification of peptides in sequence databases by peptide sequence tags.
Anal Chem. 1994 Dec 15;66(24):4390-9
Authors: Mann M, Wilm M
We demonstrate a new approach to the identification of mass spectrometrically fragmented peptides. A fragmentation spectrum usually contains a short, easily identifiable series of sequence ions, which yields a partial sequence. This partial sequence divides the peptide into three parts-regions 1, 2, and 3-characterized by the added mass m1 of region 1, the partial sequence of region 2, and the added mass m3 of region 3. We call the construct, m1 partial sequence m3, a "peptide sequence tag" and show that it is a highly specific identifier of the peptide. An algorithm developed here that uses the sequence tag to find the peptide in a sequence database is up to 1 million-fold more discriminating than the partial sequence information alone. Peptides can be identified even in the presence of an unknown posttranslational modification or an amino acid substitution between an entry in the sequence database and the measured peptide. These concepts are demonstrated with model and practical examples of electrospray mass spectrometry/mass spectrometry of tryptic peptides. Just two to three amino acid residues derived by fragmentation are enough to identify these peptides. In peptide mapping applications, even less information is necessary.
PMID: 7847635 [PubMed - indexed for MEDLINE]
Peptide mass maps: a highly informative approach to protein identification.
Anal Biochem. 1993 Nov 1;214(2):397-408
Authors: Yates JR, Speicher S, Griffin PR, Hunkapiller T
A computer searching algorithm has been used to identify protein sequences in the Protein Information Resource (PIR) database with peptide mass information (mass map) obtained from proteolytic digests of proteins analyzed by microcapillary high-performance liquid chromatography electrospray ionization mass spectrometry. A theoretical analysis of the cytochrome c family demonstrates the ability to identify protein sequences in the PIR database with a high degree of accuracy using a set of six predicted tryptic peptide masses. This method was also applied to experimentally determined peptide masses for a small GTP-binding protein, a protein from pig uterus, the human sex steroid binding protein, and a thermostable DNA polymerase. The results demonstrate that a set of observed masses which is less than 50% of the total number of predicted masses can be used to identify a protein sequence in the database. For the analysis presented in this paper, a mass matching tolerance of 1 amu is used. Under these conditions, mass maps created by fast atom bombardment mass spectrometry and matrix-assisted laser desorption time-of-flight would also be applicable. In cases where multiple matches are observed or verification of the protein identification is needed, tandem mass spectrometry sequencing can be used to establish sequence similarity.
PMID: 8109726 [PubMed - indexed for MEDLINE]
Protein identification by mass profile fingerprinting.
Biochem Biophys Res Commun. 1993 Aug 31;195(1):58-64
Authors: James P, Quadroni M, Carafoli E, Gonnet G
We have developed an algorithm for identifying proteins at the sub-microgram level without sequence determination by chemical degradation. The protein, usually isolated by one- or two-dimensional gel electrophoresis, is digested by enzymatic or chemical means and the masses of the resulting peptides are determined by mass spectrometry. The resulting mass profile, i.e., the list of the molecular masses of peptides produced by the digestion, serves as a fingerprint which uniquely defines a particular protein. This fingerprint may be used to search the database of known sequences to find proteins with a similar profile. If the protein is not yet sequenced the profile can serve as a unique marker. This provides a rapid and sensitive link between genomic sequences and 2D gel electrophoresis mapping of cellular proteins.
PMID: 8363627 [PubMed - indexed for MEDLINE]
Rapid identification of proteins by peptide-mass fingerprinting.
Curr Biol. 1993 Jun 1;3(6):327-32
Authors: Pappin DJ, Hojrup P, Bleasby AJ
Developments in 'soft' ionisation techniques have revolutionized mass-spectro-metric approaches for the analysis of protein structure. For more than a decade, such techniques have been used, in conjuction with digestion b specific proteases, to produce accurate peptide molecular weight 'fingerprints' of proteins. These fingerprints have commonly been used to screen known proteins, in order to detect errors of translation, to characterize post-translational modifications and to assign diulphide bonds. However, the extent to which peptide-mass information can be used alone to identify unknown sample proteins, independent of other analytical methods such as protein sequence analysis, has remained largely unexplored.
PMID: 15335725 [PubMed]
Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.
Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5011-5
Authors: Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C
A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.
PMID: 8506346 [PubMed - indexed for MEDLINE]
Use of mass spectrometric molecular weight information to identify proteins in sequence databases.
Biol Mass Spectrom. 1993 Jun;22(6):338-45
Authors: Mann M, Højrup P, Roepstorff P
During the last decade new ionization techniques have made it possible to measure the molecular weight of many intact proteins by mass spectrometry, and they have made it much easier to obtain a mass spectrometric peptide map of a protein. At the same time advances in protein and DNA sequencing technology are resulting in an exponential increase in the number of sequences deposited in databases. Here we investigate the possibility to use mass spectrometric data to identify proteins in databases. Searching a database by total molecular weight is found to be an easy and sometimes sufficient approach. For more specificity and for error tolerance in both the mass spectrometric data and the database information we search by partial mass spectrometric peptide map of the protein. In general, just four to six proteolytic peptides measured with a mass accuracy between 0.1 and 0.01% allow a useful search of databases such as the Protein Identification Resource (PIR). As the size of DNA and protein sequence databases grows, protein identification by partial mass spectrometric peptide maps should become increasingly powerful and may become a general method to identify and characterize proteins.
PMID: 8329463 [PubMed - indexed for MEDLINE]
The SWISS-PROT protein sequence data bank.
Nucleic Acids Res. 1991 Apr 25;19 Suppl:2247-9
Authors: Bairoch A, Boeckmann B
PMID: 2041811 [PubMed - indexed for MEDLINE]
Two dimensional gel human protein databases offer a systematic approach to the study of cell proliferation and differentiation.
Int J Dev Biol. 1989 Dec;33(4):407-16
Authors: Celis JE, Gesser B, Dejgaard K, Honoré B, Leffers H, Madsen P, Andersen A, Basse B, Celis A, Lauridsen JB
Human cellular protein databases have been established using computer-analyzed 2D gel electrophoresis. These databases, which include information on various properties of proteins, offer a global approach to the study of regulation of cell proliferation and differentiation. Furthermore, thanks to the advent of microsequencing the databases make it possible to directly link protein and DNA information.
PMID: 2701423 [PubMed - indexed for MEDLINE]
Mapping the human genome.
Lancet. 1989 Feb 4;1(8632):287
PMID: 11644426 [PubMed - indexed for MEDLINE]
Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.
Anal Chem. 1988 Oct 15;60(20):2299-301
Authors: Karas M, Hillenkamp F
PMID: 3239801 [PubMed - indexed for MEDLINE]
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
Science. 1988 Jan 29;239(4839):487-91
Authors: Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
PMID: 2448875 [PubMed - indexed for MEDLINE]
Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.
Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970-4
Authors: Aebersold RH, Leavitt J, Saavedra RA, Hood LE, Kent SB
We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from one- and two-dimensional polyacrylamide gels.
PMID: 3313383 [PubMed - indexed for MEDLINE]
Electrospray interface for liquid chromatographs and mass spectrometers.
Anal Chem. 1985 Mar;57(3):675-9
Authors: Whitehouse CM, Dreyer RN, Yamashita M, Fenn JB
PMID: 2581476 [PubMed - indexed for MEDLINE]
Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications.
J Biochem Biophys Methods. 1982 Sep;6(4):317-39
Authors: Bjellqvist B, Ek K, Righetti PG, Gianazza E, Görg A, Westermeier R, Postel W
A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described. Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacity; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strength.
PMID: 7142660 [PubMed - indexed for MEDLINE]
Fast atom bombardment: a new mass spectrometric method for peptide sequence analysis.
Biochem Biophys Res Commun. 1981 Jul 30;101(2):623-31
Authors: Morris HR, Panico M, Barber M, Bordoli RS, Sedgwick RD, Tyler A
PMID: 7306100 [PubMed - indexed for MEDLINE]
"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.
Anal Biochem. 1981 Apr;112(2):195-203
Authors: Burnette WN
PMID: 6266278 [PubMed - indexed for MEDLINE]
Quantitative comparative analysis of complex two-dimensional electropherograms.
Anal Biochem. 1979 Sep 1;97(2):210-28
Authors: Capel M, Redman B, Bourque DP
PMID: 525787 [PubMed - indexed for MEDLINE]
Quantitative analysis of two-dimensional electrophoretograms.
J Biol Chem. 1979 Aug 25;254(16):7986-98
Authors: Bossinger J, Miller MJ, Vo KP, Geiduschek EP, Xuong NH
A method for quantitative analysis of complex film density distributions in autoradiograms is described. The method is intended particularly for measuring the distribution of radioactivity among the proteins resolved by two-dimensional gel electrophoresis but should, of course, be suited to analyzing other two dimensional separations. The film density distribution is first digitized by a high speed rotating drum scanner to generate the image data array that is stored on a magnetic disk. Subsequent analysis involves: 1) data averaging, 2) detection of contours and of their locations, 3) splitting of overlapping spots, 4) conversion of film density to radioactive intensity by means of calibration films, and 5) differentiation and integration to measure the total radioactivity contained in the protein which generates a spot in the autoradiogram. The product of the analysis is a numbered contour map and a table listing coordinates and radioactivity content of each resolved spot. Coordinate transformations for comparison and matching of autoradiograms are also described. A set of utility programs print and graph the data at intermediate stages of the analysis in order to facilitate the checking of procedures and programs.
PMID: 381297 [PubMed - indexed for MEDLINE]
Modulation of protein synthesis by nerve growth factor.
J Biol Chem. 1979 Aug 25;254(16):7978-85
Authors: Garrels JI, Schubert D
The effects of nerve growth factor (NGF) and two analogs of cAMP have been studied in the PC12 line of rat pheochromocytoma cells. We have used two-dimensional gel electrophoresis to detect more than 800 proteins from control and NGF-treated cells. Of these proteins, none were qualitatively repressed in response to NGF, and no new proteins appeared after NGF treatment. Visual inspection of the gels showed that approximately 5% of the proteins were detectably increased or decreased in rate of synthesis by NGF, and each of these changes was mimicked by both cAMP analogs. The two-dimensional gel data were further analyzed by a computerized scanning system. This analysis has revealed many significant changes that are smaller than those detected by eye. Approximately 25 to 30% of the proteins analyzed were found to be altered in rate of synthesis by 30% or more. Statistical analysis has shown that the response to NGF and the response to dibutyryl cAMP are highly correlated, even down to changes as small as 30%. No proteins were found to be significantly altered by both dibutyryl cAMP and 8-bromo cAMP, but not by NGF. These results show that NGF causes only quantitative modulations of protein synthesis in PC12 cells, and these data strongly suggest that the response of PC12 cells to NGF is mediated by cAMP.
PMID: 224040 [PubMed - indexed for MEDLINE]
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7
Authors: Sanger F, Nicklen S, Coulson AR
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
PMID: 271968 [PubMed - indexed for MEDLINE]
Insertion of a rabbit beta-globin gene sequence into an E. coli plasmid.
Nucleic Acids Res. 1975 Dec;2(12):2365-78
Authors: Rougeon F, Kourilsky P, Mach B
Double stranded DNA has been synthesized in vitro from rabbit globin messenger RNA and elongated with homopolymeric dG tails. An E. coli plasmid was cleaved by EcoRI. The cohesive ends were repaired and dC tails added, to permit reconstitution of the EcoRI sites upon annealing with the dG elongated globin DNA. Transformation of E. coli with the globin-plasmid DNA hybrid has yielded a clone which harbours a recombinant plasmid (pCR1-betaG1), as demonstrated by hybridization experiments with radioactive globin cDNA. The sequence carried by the recombinant plasmid corresponds to part of the gene sequence coding for the beta chain of rabbit globin. Circular DNA of the purified recombinant plasmid exhibits sensitivity to EcoRI.
PMID: 802513 [PubMed - indexed for MEDLINE]
High resolution two-dimensional electrophoresis of proteins.
J Biol Chem. 1975 May 25;250(10):4007-21
Authors: O'Farrell PH
A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-diminsional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10 minus 4 to 10 minus 5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.
PMID: 236308 [PubMed - indexed for MEDLINE]
Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes.
Biochim Biophys Acta. 1971 Dec 28;251(3):427-34
Authors: Engvall E, Jonsson K, Perlmann P
Alkaline phosphatase from calf intestinal mucosa has been conjugated to a protein antigen, rabbit IgG. Such conjugates, prepared by glutardialdehyde, have been used in a competitive solid phase immunoassay. In this test native antigen inhibits the binding of the conjugate to homologous antibodies adsorbed to plastic tubes. Using this assay 1-100 ng/ml of the antigen could be determined.
PMID: 11452886 [PubMed - indexed for MEDLINE]
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature. 1970 Aug 15;227(5259):680-5
Authors: Laemmli UK
PMID: 5432063 [PubMed - indexed for MEDLINE]
2-dimensional resolution of plasma proteins by combination of polyacrylamide disc and gradient gel electrophoresis.
Nature. 1969 Mar 15;221(5185):1056-7
Authors: Margolis J, Kenrick KG
PMID: 5774398 [PubMed - indexed for MEDLINE]
Isoelectric focusing in polyacrylamide gel and its application to immunoglobulins.
Nature. 1968 Jul 6;219(5149):66-7
Authors: Awdeh ZL, Williamson AR, Askonas BA
PMID: 4173351 [PubMed - indexed for MEDLINE]
Isoelectric focusing in polyacrylamide gels.
Lancet. 1968 Apr 20;1(7547):847-8
Authors: Dale G, Latner AL
PMID: 4172401 [PubMed - indexed for MEDLINE]
A protein sequenator.
Eur J Biochem. 1967 Mar;1(1):80-91
Authors: Edman P, Begg G
PMID: 6059350 [PubMed - indexed for MEDLINE]