ISSN:0173-0835    

DOI:10.1002/elps.1150190602

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractAnalytical biochemistry, in particular the analysis of regulatory proteins that control biological systems and pathways, is dependent on methods of ever‐increasing sensitivity. Capillary electrophoresis (CE) has long been recognized as an ultrasensitive analytical technique. In spite of the high sensitivity, CE has not penetrated protein discovery research as a standard analytical method. In this review article we summarize recent technical developments which have significantly enhanced CE as a tool for the analysis of trace amounts of proteins. Specifically, we review recent advances in the development and application of capillary electrophoresis‐mass spectrometry (CE‐MS) and on‐line analyte concentration techniques, and introduce the emerging field of microfluidics as a front end to mass spectromet

ISSN:0173-0835    

DOI:10.1002/elps.1150190603

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractLarge‐scale DNA sequencing is creating a sequence infrastructure of great benefit to protein biochemistry. Concurrent with the application of large‐scale DNA sequencing to whole genome analysis, mass spectrometry has attained the capability to rapidly, and with remarkable sensitivity, determine weights and amino acid sequences of peptides. Computer algorithms have been developed to use the two different types of data generated by mass spectrometers to search sequence databases. When a protein is digested with a site‐specific protease, the molecular weights of the resulting collection of peptides, the mass map or fingerprint, can be determined using mass spectrometry. The molecular weights of the set of peptides derived from the digestion of a protein can then be used to identify the protein. Several different approaches have been developed. Protein identification using peptide mass mapping is an effective technique when studying organisms with completed genomes. A second method is based on the use of data created by tandem mass spectrometers. Tandem mass spectra contain highly specific information in the fragmentation pattern as well as sequence information. This information has been used to search databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (EST) sequences. The ability to search nucleotide databases is an advantage when analyzing data obtained from organisms whose genomes are not yet completed, but a large amount of expressed gene sequence is available (e.g., human and mouse). Furthermore, a strength of using tandem mass spectra to search databases is the ability to identify proteins present in fairly complex mix

ISSN:0173-0835    

DOI:10.1002/elps.1150190604

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe main factor limiting the sensitivity range for the identification of proteins isolated by two‐dimensional (2‐D) electrophoresis is sample handling: protein detection limits on the gel, losses during extraction and digestion, as well as interference of gel contaminants and detergents with the mass spectrometry (MS) detection increasing background noise. At the one hundred picomole level, losses are fairly negligible but when the amounts drop below 1 picomole (and subfemtomole peptide detection limits have been reported recently by MS), the losses become a critical point. In order to extend proteome analysis to include very low copy number proteins, methods must be developed to minimize losses and handling steps, maximize digestion and extraction yields, as well as to lower chemical noise. We present several methods that we have developed in our laboratory to: (i) increase the amount of material available in a sodium dodecyl sulfate (SDS)‐free form which does not require staining, (ii) increase protein extraction and digestion yields and lower the contamination by autoproteolytic products, and (iii) allow direct modification of the peptide mixture to generate sequence

ISSN:0173-0835    

DOI:10.1002/elps.1150190605

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractWe here describe the use of added reversed‐phase chromatographic beads to concentrate peptides from highly diluted solutions. In the procedure developed, peptide‐bead suspensions are dried under vacuum to complete dryness; peptides are subsequently eluted in a small volume of matrix‐assisted laser desorption/ionization (MALDI)‐matrix containing organic/aqueous solvent and transferred to a MALDI‐target for mass analysis. We show that by using this bead‐peptide concentration procedure, low femtomole amounts of peptides are efficiently concentrated, up to 1000 times, to volumes smaller than 0.7 μuL. We have used this concentration procedure in combination with MALDI‐post‐source decay analysis to identify subpicomole amounts of proteins present in polyacrylamide gels. Furthermore, we show that the bead‐peptide concentration method can be elegantly used to clean up samples contaminated with high concentrations of substances normally deleterious to MALDI‐mass spectrometry (MS) experiments. We have found additionally that the bead‐peptide concentration procedure can be successfully used to store low femtomole amounts of peptide for prolonged periods of time without severe losses of peptide material. This bead‐peptide concentration procedure therefore seems to be a simple and convenient step in the MALDI‐MS

ISSN:0173-0835    

DOI:10.1002/elps.1150190606

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractIdentification of proteins separated by two‐dimensional electrophoresis (2‐DE) is a necessary task to overcome the purely descriptive character of 2‐DE and a prerequisite to the construction of 2‐DE databases in proteome projects. Matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver‐stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R‐250‐stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low‐quantity proteins can be identified better starting with CBB G‐250 or Zn‐imidazol‐stained proteins. In contrast, for high‐quantity CBB R‐250‐stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium‐quantity spots after combination of tryptic di

ISSN:0173-0835    

DOI:10.1002/elps.1150190607

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractAdditional, essentially chemical, identification methods of proteins in polyacry‐lamide gel electrophoresis are described. Two cleavages of peptide bonds were used at theC‐side of aspartic acid with a 0.2% pentafluoropropionic acid (PFPA) aqueous vapor at 90° for 4–16 h, and theN‐side of serine/threonine with anS‐ethyl trifluorothioacetate vapor at 50° for 6–24 h. The products were analyzed by mass spectrometry– peptide mass fingerprinting. A new type ofC‐terminal sequencing at multisites of protein was introduced. An aqueous vapor of 90% PFPA at 90° for 2–16 h provided cleavages at theC‐side of aspartic acid and theN‐side of serine/threonine and simultaneous successive truncation at theC‐termini of the cleaved fragments. The product resulted inC‐terminal sequences at multisites in proteins by mass spectrometric analysis. The following chemical deblocking methods were used. Anhydrous hydrazine vapor at–5° for 8 h deblocked theN‐formyl group, and the vapor at 20° for 4 h deblocked pyrrolidone carboxylate.N‐acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50° for 1 h, followed by reaction withp‐suifophenylisothiocyanate at pH 6.0. These methods were applied to a variety of protein spots on polyacrylamide gels. A new stepwiseC‐terminal sequencing of protein f

ISSN:0173-0835    

DOI:10.1002/elps.1150190608

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractWe have compared several different experimental systems currently in use in our laboratory for protein identification by high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MS/MS) after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). The efficiency of peptide recovery from trypsin‐digested gel bands or electroblotted membrane slices was examined using35S‐labeled yeast proteins, and was found to be in excess of 80%. A dilution series of two standard proteins, bovine serum albumin (BSA) and carbonic anhydrase (CA), was analyzed by HPLC‐ESI‐MS/MS to determine what amount of protein could be loaded onto a gel and successfully identified, a measure we refer to as the practical detection limit. We were able to identify both standards at the 500 ng level in samples prepared from gel slices, using either a regular spray or a flow‐split microspray HPLC‐MS interface system. In samples prepared from membrane pieces, carbonic anhydrase was also identified at the 500 ng level, while bovine serum albumin could only be identified in samples of more than 1000 ng. In general, protein identification was slightly better in samples prepared from gels rather than membranes. A dilution series of lesser amounts of the same standard proteins was also analyzed using a gradient capillary LC system and we were able to successfully identify 50 ng of carbonic anhydr

ISSN:0173-0835    

DOI:10.1002/elps.1150190609

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐DE) in combination with mass spectrometry is an extremely powerful tool for characterizing complex mixtures of proteins. In many cases, the success of this approach relies upon the ability to recover peptides at high concentrations and free of interfering artifacts from in‐gel and/or on‐membrane enzymatic digests. In previous studies, we demonstrated that capillary or microcolumn ( ≤ 350 μm ID) rever‐sed‐phase high performance liquid chromatography (RP‐HPLC) is a powerful microseparation technique for proteins and peptides (Moritz, R. L. and Simpson, R. J.,J. Chromatogr.1992,599, 119–130). Here we evaluate various capillary column RP‐HPLC/mass spectrometric approaches for identifying and characterizing 2‐DE resolved proteins. For these studies, stable and efficient 0.20 mm and 0.32 mm internal diameter (ID) fused‐silica columns with hydrophilic polyvinylidene difluoride (PVDF) frits were fabricated and slurry packed with 7 μm spherical, 300 Å pore size, C8 bonded phase silica particles. We show that capillary column chromatography is a rapid and efficient desalting/concentrating (ON/OFF) technique for sample cleanup prior to protein identification by peptide‐mass fingerprinting using matrix‐assisted laser desorption ionization (MALDI)‐time‐of‐flight mass spectrometry. While marginally more peptide mass information can be obtained by stepped elution of the peptide mixture with increasing concentrations of organic solvent, best results were obtained by fractionation of the peptide mixture using a linear 60 min gradient. One salient feature of this study was the observation that, in contrast to the stepped elution and gradient approaches, the ionization of peptide T1 (m/z2402.2 SGETEDTFIADLVV(PeCys)TGQIK) was almost completely suppressed using the ON/OFF approach. Maximal amino acid sequence coverage, a necessary prerequisite for complete characterization of a protein, was accomplished using a capillary column (0.2 mm ID) directly coupled with an electro‐spray ionization (ESI) ion‐trap tandem mass spectrometer. For example, from anin situtryptic digest of α‐enolase isolated by 2‐DE from the human breast carcinoma cell line MDA‐MB231, 71% of the amino acid sequence was obtained. In addition to identifying two possibleN‐terminal acetylated α‐enolase variants, Asn153Asp and Ile152Asp/Asn153Ile, the tandem mass spectrometric data revealed the presence of a number of process‐induced modifications of α‐enolase such as methionine oxidation and cysteine amidoe‐thylationPart of this work was presented at ICES '97, International Council of Electrophoretic Soci

ISSN:0173-0835    

DOI:10.1002/elps.1150190610

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe current paradigm for protein identification using mass spectrometric derived peptide‐mass and fragment‐ion data employs computer algorithms which match uninterpreted or partially interpreted fragment‐ion data to sequence databases, both protein and translated nucleotide sequence databases. Nucleotide sequence databases continue to grow at a rapid rate for some species, providing an unsurpassed resource for protein identification in those species. Ion‐trap mass spectrometers with their ability to rapidly generate fragment‐ion spectra in a data‐dependent manner with high sensitivity and accuracy has led to their increased use for protein identification. We have investigated various parameters on a commercial ion trap‐mass spectrometer to enhance our ability to identify peptides separated by capillary reversed phase‐high performance liquid chromatography (RP‐HPLC) coupled on‐line to the mass spectrometer. By systematically evaluating the standard parameters (ion injection time and number of microscans) together with selection of multiple ions from the full mass range, improved tandem mass spectrometry (MS/MS) spectra were generated, facilitating identification of proteins at a low pmol level. Application of this technology to the identification of a standard protein and an unknown from an affinity‐enric

ISSN:0173-0835    

DOI:10.1002/elps.1150190611

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY