摘要:

AbstractMatrix‐assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin‐layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of α‐ and β‐chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one‐dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time‐lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa α‐subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)‐polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amen

ISSN:0173-0835    

DOI:10.1002/elps.1150180312

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

年代:1997

数据来源: WILEY

摘要:

AbstractWe report a rapid method for identifying proteins resolved by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) using matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS). In‐gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI‐MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in‐gel digestion were used without purification to obtain molecular weights by MALDI‐MS with α‐cyano, 4‐hydroxy‐cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detetion sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI‐MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptidematching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2‐D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low‐abundance spot was not identified by MALDI‐MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2‐D PAGE

ISSN:0173-0835    

DOI:10.1002/elps.1150180313

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

年代:1997

数据来源: WILEY

摘要:

AbstractIn peptide mass fingerprinting, there are frequently peptides whose masses cannot be explained. These are usually attributed to either a missed cleavage site during the chemical or enzymatic cutting process, the lack of reduction and alkylation of a protein, protein modifications like the oxidation of methionine, or the presence of protein post‐translational modifications. However, they could equally be due to database errors, unusual splicing events, variants of a protein in a population, or artifactual protein modifications. Unfortunately the verification of each of these possibilities can be tedious and time‐consuming. To better utilize annotated protein databases for the understanding of peptide mass fingerprinting data, we have written the program “PEPTIDE‐MASS”. This program generates the theoretical peptide masses of any protein in the SWISS‐PROT database, or of any sequence specified by the user. If the sequence is derived from the SWISS‐PROT database, the program takes into account any annotations for that protein in order to generate the peptide masses. In this manner, the user can obtain the predicted masses of peptides from proteins which are known to have signal sequences, propeptides, transit peptides, simple post‐translational modifications, and disulfide bonds. Users are also warned if any peptide masses are subject to change from protein iso‐forms, database conflicts, or an mRNA splicing variation. The program is freely accessible to the scientific communityviathe ExPASy World Wide Web server, at the URL address:www.expasy.

ISSN:0173-0835    

DOI:10.1002/elps.1150180314

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

年代:1997

数据来源: WILEY

摘要:

AbstractTwo‐dimensional (2‐D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first‐dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2‐D gel electrophoresis with mass spectrometry to identify interferon‐γ‐ (IFN) and tumor necrosis factor (TNF)‐regulated proteins in ME‐180 cervical carcinoma cells. Three cytokine‐regulated proteins have been identified, using imidazole‐zinc‐stained preparative IPG 2‐D gels and in‐gel tryptic digestion followed by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry for determination of peptide masses and sequences: (1) triosephosphate isomerase, a glycolytic pathway enzyme, (2) proteasome subunit C3, which is important in protein degradation, and (3) Ran, a GTP‐binding protein important in cell cycle regulation, protein import into the nucl

ISSN:0173-0835    

DOI:10.1002/elps.1150180315

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

年代:1997

数据来源: WILEY

摘要:

AbstractProtein extract from yeast cells growing exponentially in saline medium was separated by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), with the separation in the first dimension on a wide range immobilized pH (3–10) gradient. From one preparative 2‐D gel a number of previously identified proteins were used as test material for our initial matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) efforts on large scale rapid protein spot identification. Sample preparation via in‐gel trypsin digestion was slightly modified to be compatible to MS analysis, andviathis modified procedure MS generated peptide mass profiles could, in most cases with good precision, identify the protein in question. Preferential ionization was tested on a yeast aldehyde dehydrogenase (ALD7), and it was shown that the ionization of some peptides was clearly suppressed by the presence of others. Roughly 50% of the observed peptide masses was found by the search routines in the database, and the mass measurement accuracy of the peptides was within 0.5 Da. Silver‐stained gels could be used with good results for the generation of peptides to be analyzed by MALDI‐MS. For one of the 2‐D resolved proteins, glycerol 3‐phosphatase (GPP1), the post‐source decay (PSD) spectrum proved cr

ISSN:0173-0835    

DOI:10.1002/elps.1150180316

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

年代:1997

数据来源: WILEY

摘要:

AbstractThe 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 MA

ISSN:0173-0835    

DOI:10.1002/elps.1150180317

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

年代:1997

数据来源: WILEY

摘要:

AbstractRecently the determination of the genome sequences of three procaryotes (Haemophilus influenzae, Methanococcus jannaschii and Mycoplasma genitalium) as well as the first eucaryotic genome (Saccharomyces cerevisiae) were completed. Between 40–60% of the genes were found to code for proteins to which no function could be assigned. We describe an approach which combines proteome analysis (mapping of expressed proteins isolated by two‐dimensional polyacrylamide gel electrophoresis to the genome) with genetic manipulations to study the complex pattern of protein regulation occurring inEscherichia coliin response to sulfate starvation. We have previously described the upregulation of eight spots on two‐dimensional (2‐D) gels in response to sulfate starvation and the assignment of six of these to entries in theE. coligenome sequence (Quadroniet al., Eur. J. Biochem.1996,239, 773–781). Here we describe the identification of the remaining two proteins which are encoded in a sulfate‐controlled operon in the 21.5′ region of theE. coligenome. Upregulated protein spots were cut from multiple 2‐D gels collected and run on a modified funnel gel to concentrate the proteins and remove the sodium dodecyl sulfate before digestion. The peptide masses obtained from the digests were used to search the SwissProt database or a six‐frame translation of the EMBL DNA database using a peptide mass fingerprinting algorithm. A digest can be reanalyzed after deuterium exchange to obtain a second, orthogonal data set to increase the confidence level of protein identification. The digests of the remaining unidentified proteins were used for peptide fragment generation using either post‐source decay in a matrix‐assisted laser desorption ionization (MALDI) time‐of‐flight mass spectrometer or collision‐induced dissociation (CID) coupled mass specrometry (MS/MS) with triple stage quadrupole or ion trap mass spectrometers. The spectra were used as peptide fragment fingerprints to search the S

ISSN:0173-0835    

DOI:10.1002/elps.1150180318

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

年代:1997

数据来源: WILEY

摘要:

AbstractThe amino acid composition (AAC)versusthe protein identity (PI) method was used for establishment of the identities of proteins from bovine brain and kidneys which were prefractionated on a CM52 cation exchanger and by preparative flat‐bed isoelectric focusing. Established identities of proteins whose AACs converge with those of other members of their proper superfamily are reliable. Groups of convergent AACs can be extracted from protein databases using the standard root‐mean‐square rule (Rmsd) with measures the difference between the AAC of chosen proteinversusthose in the database. Convergence of AACs of proteins is dependent on several factors such as the upper limit of Rmsd, the limits of variations of molecular mass (m) and isoelectric point (pI), the number of proteins with similar AACs present in protein databases, and the domain structure of proteins. AACs of many proteins remain unique if the Rmsd is maintained with 1.5–1.0 withm± 3kDA and pI± 4. Certain groups of multidomain proteins have quasi‐unique AACs only if the Rmsd is restrained to a value within 1.0 and 0.7. Convergence of AACs of certain groups of proteins may indicate that a common biological function exists for some members of each group. The AAC‐PI method may become an additional search tool for prot

ISSN:0173-0835    

DOI:10.1002/elps.1150180319

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

年代:1997

数据来源: WILEY

摘要:

AbstractPreparative two‐dimensional polyacrylamide electrophoresis (2‐D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2‐D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious ‘trains’ of spots which differ in pIand/or apparent molecular mass. These are usually isoforms of the same protein and result from post‐translational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2‐D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2‐D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2‐D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2‐D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single

ISSN:0173-0835    

DOI:10.1002/elps.1150180320

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

年代:1997

数据来源: WILEY

摘要:

AbstractScientists around the world often work on similar data so the need to share results and compare data arises periodically. We describe a method of comparing two two‐dimensional (2‐D) protein gels of similar samples created in different laboratories to help identify or suggest protein spot identification. Now that 2‐D gels and associated databases frequently appear on the Internet, this opens up the possibility of visually comparing one's own experimental 2‐D gel image data with data from another gel in a remote Internet database. In general, there are a few ways to compare images: (i) slide one gel (autoradiograph or stained gel) over the other while back‐illuminated, or (ii) build at 2‐D gel computer database from both gels after scanning and analyzing these gels. These are impractical since in the first case the gel from the Internet database is not locally available. In the second, the costs of building a multi‐gel database solely to answer the question of whether a spot is the same spot may be excessive if only a single visual comparison is needed. We describe a distributed gel comparison program (URL:www‐lmmb.ncifcrf.gov/flicker) which runs on any World Wide Web (WWW) connected computer and is invoked from a Java‐capable web browser. One gel image is read from any Internet 2‐D gel database (e.g.SWISS‐2DPAGE) and the other may reside on the investigator's computer. Images may be more easily compared by first applying spatial warping or other transforms interactively on the user's computer. First, regions of interest are “landmarked” with several corresponding points in each gel image, then one gel image is warped to the geometry of the other. As the two gels are rapidly alternated, or flickered, in the same window, the user can slide one gel past the other to visually align corresponding spots by matching local morphology. This flicker‐comparison technique may be applied to analyzing other types of one‐dimensio

ISSN:0173-0835    

DOI:10.1002/elps.1150180321

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

年代:1997

数据来源: WILEY