Micro-scale peptide mapping method for identification of variant proteins

A micro-scale (for protein of the order of 10^–9 mole) peptide mapping method in thin-layer cellulose is reported. Peptide maps of tryptic digests from glucagon, from normal and variant -chains of human hemoglobin, from human glucose 6-phosphate dehydrogenase, and from human phosphoglycerate kinase showed satisfactory resolution; thus this method can be used for characterizing the structure of variant enzymes and proteins of 10^–9 molar quantity.This research was supported by research grant GM 15253 from the National Institutes of Health.     

Enhancing peptide identification confidence by combining search methods

Confident peptide identification is one of the most important components in mass-spectrometry-based proteomics. We propose a method to properly combine the results from different database search methods to enhance the accuracy of peptide identifications. The database search methods included in our analysis are SEQUEST (v27 rev12), ProbID (v1.0), InsPecT (v20060505), Mascot (v2.1), X! Tandem (v2007.07.01.2), OMSSA (v2.0) and RAId_DbS. Using two data sets, one collected in profile mode and one collected in centroid mode, we tested the search performance of all 21 combinations of two search methods as well as all 35 possible combinations of three search methods. The results obtained from our study suggest that properly combining search methods does improve retrieval accuracy. In addition to performance results, we also describe the theoretical framework which in principle allows one to combine many independent scoring methods including de novo sequencing and spectral library searches. The correlations among different methods are also investigated in terms of common true positives, common false positives, and a global analysis. We find that the average correlation strength, between any pairwise combination of the seven methods studied, is usually smaller than the associated standard error. This indicates only weak correlation may be present among different methods and validates our approach in combining the search results. The usefulness of our approach is further confirmed by showing that the average cumulative number of false positive peptides agrees reasonably well with the combined E-value. The data related to this study are freely available upon request.     

Evaluation of proteome reference maps for cross-species identification of proteins by peptide mass fingerprinting

We tested whether proteome reference maps established for one species can be used for cross-species protein identification by comparing two-dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species. First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability. Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross-species similarities. In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities. However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species. Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species-specific protein identification than N-terminal sequencing. While peptide masses were found to be unreliable for cross-species protein identification, we present useful criteria to determine confident matching against species-specific expressed sequence tag databases. In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protein identification.     

Rapid identification of probiotic lactobacillus biosurfactant proteins by ProteinChip tandem mass spectrometry tryptic peptide sequencing

A novel ProteinChip-interfaced tandem mass spectrometer was employed to identify collagen binding proteins from biosurfactant produced by Lactobacillus fermentum RC-14. On-chip tryptic digestion of the captured collagen binding proteins resulted in rapid sequence identification of five novel tryptic peptide sequences via collision-induced dissociation tandem mass spectrometry.     

Selection of the peptide mass tolerance value for protein identification with peptide mass fingerprinting

Peptide mass fingerprinting (PMF) is widely used for protein identification while studying proteome via time-of-flight mass spectrometer or via 1D or 2D electrophoresis. Peptide mass tolerance indicating the fit of theoretical peptide mass to an experimental one signifcantly influences protein identification. The role of peptide mass tolerance could be estimated by counting the number of correctly identified proteins for the reference set of mass spectra. The reference set of 400 Ultraflex (Bruker Daltonics, Germany) protein mass spectra was obtained for liver microsomes slices hydrolyzed via 1D gel electrophoresis. Using a Mascot server for protein identification, the peptide mass tolerance value varied within 0.02–0.40 Da with a step of 0.01 Da. The number of identified proteins changed up to 10 times depending on the tolerance. The maximal number of identified proteins was reported for the tolerance value of 0.15 Da (120 ppm) known to be 1.5–2-fold higher than the recommended values for such a type of mass spectrometer. The software program PMFScan was developed to obtain the dependence between the number of identified proteins and the tolerance values.     

Selection of the peptide mass tolerance value for the protein identification with peptide mass fingerprinting

Peptide mass-fingerprint is widely used for protein identification while studying proteome with the use of 1D or 2D electrophoresis. Peptide mass tolerance indicates the fit of theoretical peptide mass with the experimental measurements, and choice of this parameter sufficiently influences the protein identification. The role of peptide mass tolerance was estimated by counting the number of identified proteins for the reference set of mass-spectra. The reference set of 400 Ultraflex (Bruker Daltonics, Germany) mass-spectra was obtained for the slices of 1D gel of liver microsomes. Using Mascot server for protein identification, the peptide mass tolerance value was varied in the range from 0.02 to 0.40 Da with a step 0.01 Da. Depending on the tolerance the number of identified protein changes up to 10 times. Maximal number of identified proteins was reported for the tolerance value of 0.15 Da (120 ppm), which is 1.5 - 2 times higher than the recommended values for such type of mass-spectrometers. The software program PMFScan was developed to obtain the dependence of number of identified proteins of the tolerance values.     

Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry

Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel.     

Rapid identification of DNA-binding proteins by mass spectrometry.

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.     

Artifacts and unassigned masses encountered in peptide mass mapping

In peptide mass mapping of isolated proteins, a significant number of the observed mass spectral peaks are often uninterpreted. These peaks derive from a number of sources: errors in the genome that give rise to incorrect peptide mass predictions, undocumented post-translational modifications, sample handling-induced modifications, contaminants in the sample, non-standard protein cleavage sites, and non-protein components of the sample. In a study of the stalk organelle of Caulobacter crescentus, roughly one-third (782/2215) of all observed masses could not be assigned to the proteins identified in the gel spots (Karty et al., J. Proteome Res., 1 (2002) 325). By interpreting these masses, this work illuminates a number of phenomena that may arise in the course of peptide mass mapping of electrophoretically separated proteins and presents results from a number of related studies.     

C-terminal peptide identification by fast atom bombardment mass spectrometry.

A previously described technique [Rose, Simona, Offord, Prior, Otto & Thatcher (1983) Biochem. J. 215, 273-277] permits the identification of the C-terminal peptide of a protein as the only peptide that does not incorporate any 18O upon partial enzymic hydrolysis in 18O-labelled water. Formation of chemical derivatives followed by combined g.l.c.-m.s. was used in this earlier work. We now describe the isolation from protein digests, by reversed-phase h.p.l.c., of labelled and unlabelled polypeptides and their direct analysis by fast atom bombardment mass spectrometry. Under the conditions used, the 18O label is retained throughout the separation and analysis, thus permitting assignments of C-terminal peptides to be made. Enzyme-catalysed exchange of label into the terminal carboxy group was found to occur in some cases without hydrolysis of a peptide bond. This effect, which may be exploited to prepare labelled peptides, does not prevent application of the method (two separate digests must then be used). We have applied our method to the analysis of enzymic partial hydrolysates of glucagon, insulin and of several proteins produced by expression of recombinant DNA.     

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.     

Enzymatic cleavage and HPLC peptide mapping of proteins

Detailed procedures are described for successfully digesting reasonably small quantities (i.e., usually >10 pmol) of proteins with a variety of proteases and for then isolating the resulting peptides by reversephase HPLC. Since sodium dodecyl sulfate-polyacrylamide, gel electrophoresis (SDS-PAGE) appears to be the current method of choice for final purification of proteins for structural analysis, special attention is given to carrying out in-gel proteolytic digests on SDS-PAGE-separated proteins that have usually been stained with Coomassie Blue. A compilation of data from nearly 200 “unknown” samples is used to help provide realistic expectations with respect to the results that are likely to be obtained from carrying out in-gel proteolytic digests on large numbers of proteins.     

Protein separation and characterization by np-RP-HPLC followed by intact MALDI-TOF mass spectrometry and peptide mass mapping analyses

Because of their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of nonporous-reversed-phase (np-RP)-HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP-HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF-MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF-MS was in the 10-50 fmol level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall, we demonstrate that np-RP-HPLC followed by MALDI-TOF-MS allows for rapid, sensitive, and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information.     

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting

We compared trysin-digested protein samples desalted by ZipTip(C18) reverse-phase microcolumns with on-plate washing of peptides deposited either on paraffin-coated plates (PCP), Teflon-based AnchorChip plates, or stainless steel plates, before analysis by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Trypsinized bovine serum albumin and ovalbumin and 16 protein spots extracted from silver-stained two-dimensional gels of murine C(2)C(12) myoblasts or human leukocytes, prepared by the above two methods, were subjected to MALDI on PCP, AnchorChip plates, or uncoated stainless steel plates. Although most peptide mass peaks were identical regardless of the method of desalting and concentrating of protein samples, samples washed and concentrated by the PCP-based method had peptide peaks that were not seen in the samples prepared using the ZipTip(C18) columns. The mass spectra of peptides desalted and washed on uncoated stainless steel MALDI plates were consistently inferior due to loss of peptides. Some peptides of large molecular masses were apparently lost from samples desalted by ZipTip(C18) microcolumns, thus diminishing the quality of the fingerprint needed for protein identification. We demonstrate that the method of washing of protein samples on paraffin-coated plates provides an easy, reproducible, inexpensive, and high-throughput alternative to ZipTip(C18)-based purification of protein prior to MALDI-TOF-MS analysis.     

A fast coarse filtering method for peptide identification by mass spectrometry

MOTIVATION: We reformulate the problem of comparing mass-spectra by mapping spectra to a vector space model. Our search method leverages a metric space indexing algorithm to produce an initial candidate set, which can be followed by any fine ranking scheme. RESULTS: We consider three distance measures integrated into a multi-vantage point index structure. Of these, a semi-metric fuzzy-cosine distance using peptide precursor mass constraints performs the best. The index acts as a coarse, lossless filter with respect to the SEQUEST and ProFound scoring schemes, reducing the number of distance computations and returned candidates for fine filtering to about 0.5% and 0.02% of the database respectively. The fuzzy cosine distance term improves specificity over a peptide precursor mass filter, reducing the number of returned candidates by an order of magnitude. Run time measurements suggest proportional speedups in overall search times. Using an implementation of ProFound's Bayesian score as an example of a fine filter on a test set of Escherichia coli protein fragmentation spectra, the top results of our sample system are consistent with that of SEQUEST.     

Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping

Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3–10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight > 10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots.     

Protease peptide mapping of affinity-labeled rat pancreatic cholecystokinin-binding proteins

Affinity-labeling probes with sites of cross-linking distributed along the ligand have been used to biochemically characterize the pancreatic cholecystokinin (CCK) receptor. Probes with photolabile sites spanning the receptor-binding domain have labeled a Mr = 85,000-95,000 plasma membrane protein, while a probe cross-linked via the amino terminus of CCK-33, far removed from the carboxyl-terminal receptor-binding domain, has labeled a distinct Mr = 80,000 protein. In this work, protease peptide mapping of the pancreatic proteins labeled by each of these probes has been performed to gain insight into the identities of the bands and to define domains of the labeled proteins. Photolabile decapeptide probes with sites of cross-linking at the amino terminus, mid region, and carboxyl terminus of the receptor-binding domain each labeled a Mr = 85,000-95,000 glycoprotein with a Mr = 42,000 core protein and similar Staphylococcus aureus V8 protease peptide maps. This confirms that each probe labels the same binding protein and the same domain of that protein. Serial slices through the broad labeled band were separately deglycosylated and protease-treated, demonstrating a single protein core with differential glycosylation. The CCK-33-based probe, however, labeled predominantly two proteins, one having similar sizes in its native and deglycosylated forms to that labeled by the decapeptide probes and a distinct Mr = 80,000 protein. Of note, the peptide map of the protein believed to be the same as that labeled by the shorter probes was different, suggesting that this probe labeled the binding subunit at a site distinct from that which was labeled by the short probes.     

Defining the limits of diving biochemistry in marine mammals

The field of marine mammal diving biochemistry was essentially untouched when Peter Hochachka turned his attention to it in the mid-1970s. Over the next 30 years, his work followed three main themes in this area: first, most biologists at that time supported the theory that diving mammals utilized enhanced metabolic pathways for hypoxic energy production (glycolysis to lactate) and reduced their metabolic rate while diving. Peter began his work on potential hypoxic adaptations in marine mammals by working out the details of how these pathways would be regulated. By the 1980s, he started to ask how diving mammals balanced the increased demands of exercise with the apparently conflicting demands to reduce aerobic metabolism while exercising underwater. By the 1990s, his work involved complex models of the interplay between the neural, hormonal, behavioral and evolutionary components of diving biochemistry and animal exercise. From a comparative approach, he excelled at bringing themes of hypoxic adaptation from many different types of animals to the field of diving mammal biochemistry. This review traces the history of Peter Hochachka's work on diving biochemistry from the perspective of those of us who spent time with him both inside the laboratory and outside in the field from Antarctica to Iceland.