Protein identification methods in proteomics

A combination of high-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis, highly sensitive biological mass spectrometry, and the rapidly growing protein and DNA databases has paved the way for high-throughput proteomics. This review concentrates on protein identification. We first discuss the use of protein electroblotting and Edman sequencing as tools for de novo sequencing and protein identification. In the second part, we highlight matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as one of the main contemporary analytical methods for linking gel-separated proteins to entries in sequence databases. In this context we describe the two main MALDI-MS-based identification methods: (i) peptide mass fingerprinting, and (ii) post-source decay (PSD) analysis. In the last part, we briefly emphasize the importance of sample preparation for obtaining highly sensitive and high-quality MALDI-MS spectra.     

Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.     

Modeling peptide mass fingerprinting data using the atomic composition of peptides

The peptide mass fingerprinting technique is commonly used for identifying proteins analyzed by mass spectrometry (MS) after enzymatic digestion. Our goal is to build a theoretical model that predicts the mass spectra of such digestion products in order to improve the identification and characterization of proteins using this technique. We present here the first step towards a full MS model. We have modeled MS spectra using the atomic composition of peptides and evaluated the influence that this composition may have on the MS signals. Peptides deduced from the SWISS-PROT protein sequence database were used for the calculation. To validate the model, the variability of the peptide mass distribution in SWISS-PROT was compared to two theoretical, randomly generated databases. Functions have been built that describe the behavior of the isotopic distribution according to the mass of peptides. The variability of these functions was analyzed. In particular, the influence of sulfur was studied. This work, while representing only a first step in the construction of an MS model, yields immediate practical results, as the new isotopic distribution model significantly improves peak detection in MS spectra used by protein identification algorithms.     

Efficient peptide mapping and its application to identify embryo proteins in rice proteome analysis

Using direct N-terminal analysis, only 31 N-terminally unblocked proteins out of 100 rice embryo proteins could be identified. To obtain protein sequence information for the remaining 69 blocked proteins, we developed a simple, efficient and rapid method. Using this method, we determined the peptide maps of 20 proteins per day in 10 pmol amounts. Applying this method to rice proteome analysis, we determined the internal sequences of all 69 blocked proteins. A total of 28 proteins out of 100 analyzed showed sequence similarity to the proteins with known functions in the SWISS-PROT and NCBI databases. Alternatively, we also used peptide mass fingerprinting determined by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to identify the rice proteins separated by two-dimensional electrophoresis (2-DE). Although peptide-mass fingerprinting is a high-throughput method, we could not easily identify all the rice proteins or genes by this method, because the complete database information on rice, is not yet available and many proteins are post-translationally modified. Therefore, at present, the improved peptide mapping method as we report here is considered to be very useful in rice proteome analysis, especially for blocked proteins.     

Improving protein identification from peptide mass fingerprinting through a parameterized multi-level scoring algorithm and an optimized peak detection

We have developed a new algorithm to identify proteins by means of peptide mass fingerprinting. Starting from the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectra and environmental data such as species, isoelectric point and molecular weight, as well as chemical modifications or number of missed cleavages of a protein, the program performs a fully automated identification of the protein. The first step is a peak detection algorithm, which allows precise and fast determination of peptide masses, even if the peaks are of low intensity or they overlap. In the second step the masses and environmental data are used by the identification algorithm to search in protein sequence databases (SWISS-PROT and/or TrEMBL) for protein entries that match the input data. Consequently, a list of candidate proteins is selected from the database, and a score calculation provides a ranking according to the quality of the match. To define the most discriminating scoring calculation we analyzed the respective role of each parameter in two directions. The first one is based on filtering and exploratory effects, while the second direction focuses on the levels where the parameters intervene in the identification process. Thus, according to our analysis, all input parameters contribute to the score, however with different weights. Since it is difficult to estimate the weights in advance, they have been computed with a generic algorithm, using a training set of 91 protein spectra with their environmental data. We tested the resulting scoring calculation on a test set of ten proteins and compared the identification results with those of other peptide mass fingerprinting programs.     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell--the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated.     

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.     

Sequit: software for de novo peptide sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

Peptide sequencing by mass spectrometry is gaining increasing importance for peptide chemistry and proteomics. However, available tools for interpreting matrix-assisted laser desorption/ionization post-source decay (MALDI-PSD) mass spectra depend on databases, and identify peptides by matching experimental data with spectra calculated from database sequences. This severely obstructs the identification of proteins and peptides not listed in databases or of variations, e.g. mutated proteins. The development of a new computer program for database-independent peptide sequencing by MALDI-PSD mass spectrometry is reported here. This computer program was validated by the determination of the correct sequences for various peptides including sequences listed in the sequence databases, but also for peptides that deviate from database sequences or are completely artificial. This strategy should substantially facilitate the identification of novel or variant peptides and proteins, and increase the power of MALDI-PSD analyses in proteomics.     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell – the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated. Received: 16 December 1999 / Accepted: 17 December 1999     

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.     

Characterization of ribosomal proteins as biomarkers for matrix-assisted laser desorption/ionization mass spectral identification of Lactobacillus plantarum

For rapid identification of bacteria by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a bioinformatics approach using ribosomal subunit proteins as biomarkers has been proposed. This method compares the observed masses for biomarkers with calculated masses as predicted from the amino acid sequences registered on protein databases. To evaluate this approach, the expressed ribosomal proteins of a genome-sequenced bacterium, Lactobacillus plantarum NCIMB 8826, were characterized as a model sample. The protein expression of 42 ribosomal subunit proteins, together with 10 ribosome-associated proteins in the isolated ribosome fraction, was confirmed through two-dimensional gel electrophoresis combined with peptide mass fingerprinting. The observed masses of the proteins in the isolated ribosome fraction were then determined by MALDI-MS. We preliminarily selected 44 biomarkers whose observed masses were matched with the calculated masses predicted from the amino acid sequence registered in the protein databases by considering N-terminal methionine loss only. Of these, the finally selected reliable biomarkers were 34 proteins including 31 ribosomal subunit proteins and 3 ribosome-associated proteins that could be observed in the MALDI mass spectra of the cell lysate sample. These biomarkers were usable in MALDI-MS characterization of two industrial L. plantarum cultures.     

Method for differential detection and identification of components in protein mixtures analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We demonstrate that the semi-quantitative information in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of tryptically digested protein mixtures can, via a systematic statistical approach, be utilized for the identification of a protein present in different concentrations in two samples. Multiple mass spectra were acquired from a series of tryptically digested test samples in which the concentration of one protein was varied and the concentrations of three other proteins were held constant. The mass spectra were subjected to soft independent modeling of class analogy (SIMCA) analysis assuming that spectra originating from two different samples belonged to different data classes. The SIMCA analysis yielded information on which individual m/z values discriminate between two classes. Protein identification by proteolytic peptide mass fingerprinting was performed with different numbers of mass values in the fingerprint according to the discriminatory information, beginning with the mass corresponding to the best discrimination, followed by the best together with the second best, etc. By using the Probity algorithm, which computes the statistical significance of each identification result, we demonstrate that the first protein identified at a desired significance level (0.001) is the protein that was present in a different concentration in the two samples. Differential analysis of expression is often performed by comparing 2D-gel-spot intensities followed by mass spectrometric identification of the respective protein in each spot that differs. The method presented here has the potential to allow identification of the protein component that differs in cases where a gel-spot is poorly resolved and contains several proteins.     

Towards a human repertoire of monocytic lysosomal proteins

The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well-known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins.     

Automatic poisson peak harvesting for high throughput protein identification

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel-separated bovine serum albumin, and with Escherictia coli proteins prepared by two-dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.     

Data requirements for protein identification using chemically-assisted fragmentation and tandem mass spectrometry

Many laboratories identify proteins by searching tandem mass spectrometry data against genomic or protein sequence databases. These database searches typically use the measured peptide masses or the derived peptide sequence and, in this paper, we focus on the latter. We study the minimum peptide sequence data requirements for definitive protein identification from protein sequence databases. Accurate mass measurements are not needed for definitive protein identification, even when a limited amount of sequence data is available for searching. This information has implications for the mass spectrometry performance (and cost), data base search strategies and proteomics research.     

Effect of sodium dodecyl sulfate micelles on peptide mass fingerprinting by matrix-assisted laser desorption/ionization mass spectrometry

Here we have examined the effect of sodium dodecyl sulfate (SDS) at various concentrations on matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting experiments. Several model proteins were digested with trypsin and then various amounts of SDS were added prior to MALDI mass spectrometry. Evaluation of the data was made by calculating the amino acid sequence coverage within each analysis. It was found that addition of 0.1-0.3% w/v SDS prior to MALDI analysis results in an increase in the number of tryptic peptides detected thereby improving the total sequence coverage of the analysis. The use of SDS at concentrations near its critical micelle concentration can improve sequence coverage from MALDI peptide mass fingerprinting analyses allowing for increased confidence in protein identification or additional opportunities to identify putative regions of posttranslational modification.     

CHASE, a charge-assisted sequencing algorithm for automated homology-based protein identifications with matrix-assisted laser desorption/ionization time-of-flight post-source decay fragmentation data

We describe CHASE, a novel algorithm for automated de novo sequencing based on the mass spectrometric (MS) fragmentation analysis of tryptic peptides. This algorithm is used for protein identification from sequence similarity criteria and consists of four steps: (1) derivatization of tryptic peptides at the N-terminus with a negatively charged reagent; (2) post-source decay (PSD) fragmentation analysis of peptides; (3) interpretation of the mass peaks with the CHASE algorithm and reconstruction of the amino acid sequence; (4) transfer of these data to software for protein identifications based on sequence homology (Basic Local Alignment Search Tool, BLAST). This procedure deduced the correct amino acid sequence of tryptic peptide samples and also was able to deduce the correct sequence from difficult mass patterns and identify the amino acid sequence. This allows complete automation of the process starting from MS fragmentation of complex peptide mixtures at low concentration (e.g. from silver-stained gel bands) to identification of the protein. We also show that if PSD data are collected in a single spectrum (instead of the segmented mode offered by conventional matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation), the complete workflow from MS-PSD data acquisition to similarity-based identification can be completely automated. This strategy may be applied to proteomic studies for protein identification based on automated de novo sequencing instead of MS or tandem MS patterns. We describe the Charge Assisted Sequencing Engine (CHASE) algorithm, the working protocol, the performance of the algorithm on spectra from MALDI-TOFMS and the data comparison between a TOF and a TOF-TOF instrument.     

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

A simple mass spectrometric method to sequence a recombinant phosphoenolpyruvate carboxykinase of known structure and a novel variant of unknown structure isolated from Anaerobiospirillum succiniciproducens and Actinobacillus succinogenes 130Z, respectively, was evaluated. The proteolytic digests of the proteins were each chemically derivatized at the N-terminus by addition of a tris(trimethoxyphenyl)phosphoniumacetyl (TMPP(+)-Ac) group to produce peptides with a fixed positive charge. The derivatized digests were then partially separated by reversed-phase high-performance liquid chromatography. The fractions collected were subjected to matrix-assisted laser desorption/ionization post-source decay (MALDI/PSD) mass spectrometric analysis. The resulting spectra are sufficiently simple to allow the sequence to be read directly without extensive interpretation. This is in contrast to spectra of underivatized peptides obtained by MALDI/PSD or conventional tandem mass spectrometry, where full sequence interpretation can be challenging. Aided with a set of very simple established rules, it was shown that the sequence of TMPP(+)-Ac derivatives can be derived strictly from predictable fragment ion series. In most cases, this is sufficient to determine extensive, unambiguous, peptide sequences de novo. The partial sequence (35%) of the unknown phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes 130Z was obtained entirely by the mass spectrometric method evaluated here, which provided the basis for evaluating homology and for the design of oligonucleotide probes for cloning the corresponding gene.     

De novo sequencing of tryptic peptides sulfonated by 4-sulfophenyl isothiocyanate for unambiguous protein identification using post-source decay matrix-assisted laser desorption/ionization mass spectrometry

A simple method of solid-phase derivatization and sequencing of tryptic peptides has been developed for rapid and unambiguous identification of spots on two-dimensional gels using post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The proteolytic digests of proteins are chemically modified by 4-sulfophenyl isothiocyanate. The derivatization reaction introduces a negative sulfonic acid group at the N-terminus of a peptide, which can increase the efficiency of PSD fragmentation and enable the selective detection of only a single series of fragment ions (y-ions). This chemically assisted method avoids the limitation of high background normally observed in MALDI-PSD spectra, and makes the spectra easier to interpret and facilitates de novo sequencing of internal fragment. The modification reaction is conducted in C(18) microZipTips to decrease the background and to enhance the signal/noise. Derivatization procedures were optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. The MALDI-PSD mass spectra of two model peptides and their sulfonated derivatives are compared. For some proteins unambiguous identification could be achieved by MALDI-PSD sequencing of derivatized peptides obtained from in-gel digests of phosphorylase B and proteins of hepatic satellite cells (HSC).