A typical approach to protein identification in our laboratory is outlined in Fig. 8. Protein from a polyacrylamide gel is excised and then in-gel digested with trypsin by the method of Wilm et al. (170). Following peptide extraction from the gel, we purify the peptides on Poros R2 (149, 169) in microcapillary tubes by using the method described on the website http://www.protana.com/products/applicationnotes/purification/default.asp. We use the API QSTAR Pulsar mass spectrometer (AB/MDS-SCIEX) with nanospray ionization to obtain an MS scan of the peptide mixture. From the MS scan, a peptide ion is selected for MS/MS based on its signal strength and charge state, which allow it to be distinguished from the background ions. In nanospray ionization, most peptide ions are either doubly or triply charged whereas the background ions are singly charged. This peptide ion is also known as the parent ion. MS/MS of a parent ion is performed, and amino acid sequence information for the peptide is obtained. As shown in Fig. 8, a single peptide was sequenced and found to match rhoptry-associated protein 2 (RAP-2) from Plasmodium falciparum. Since matching multiple peptides to a protein increases the confidence of identification (106), we typically sequence several peptides for each sample. For RAP-2, a total of four peptides were found to match the protein. Because the staining intensity on gels is not always a good indicator of the signal obtained by MS and because gel bands often contain protein mixtures, additional criteria can aid in protein identification. For example, if the major protein excised from the gel was 50 kDa, does the protein identified match in molecular mass? Is the protein from the expected species? If a protein is isolated from a 2-D gel, does it match the expected isoelectric point as exhibited on the gel?

View larger version (39K): [in this window] [in a new window]   FIG. 8. Protein identification by MS/MS. (A) Protein from P. falciparum was resolved on a one-dimensional polyacrylamide gel, excised, and in-gel digested with trypsin. The resulting peptides were ionized by electrospray and analyzed by a Quadrupole-TOF mass spectrometer. (B) The MS spectrum produced was scanned, and a parent ion of 678.8 was selected for fragmentation. (C) Enlargement of the parent ion peak at 678 shown in panel B. The multiplet of peaks is due to the contribution in mass from the naturally occurring isotope 13C. A mass difference between the peaks of 0.5 Da indicates that the peptide is doubly charged. (D) MS/MS scan of the 678 parent ion and analysis of the daughter ions produced. All y-ions (except for y-11) produced from fragmentation of the peptide are shown. (E) Identification of rhoptry-associated protein-2 using BioAnalyst software (Applied Biosystems, Foster City, Calif.).

Databases allow protein structural information harvested from Edman sequencing or MS to be used for protein identification. The goal of database searching is to be able to quickly and accurately identify large numbers of proteins (132). The success of database searching depends on the quality of the data obtained in the mass spectrometer, the quality of the database searched, and the method used to search the database. What is the best way to identify an unknown protein? What type of database search engine should be used?

Peptide mass fingerprinting database searching. One method of protein identification is peptide mass fingerprinting (77, 79, 102, 125, 175). In this method, the masses of peptides obtained from the proteolytic digestion of an unknown protein are compared to the predicted masses of peptides from the theoretical digestion of proteins in a database (Fig. 9). If enough peptides from the real mass spectrum and the theoretical one overlap, a protein identification can be made. The principal advantage of peptide mass fingerprinting is speed. The analysis and database search can be fully automated.

View larger version (21K): [in this window] [in a new window]   FIG. 9. Strategy of protein identification by peptide mass fingerprinting. (A) The unknown protein is excised from a gel and converted to peptides by the action of a specific protease. The mass of the peptides produced is then measured in a mass spectrometer. (B) The mass spectrum of the unknown protein is searched against theoretical mass spectra produced by computer-generated cleavage of proteins in the database.

 
The single biggest disadvantage of peptide mass fingerprinting is ambiguity in protein identification. This is because of peptide mass redundancy. For example, a peptide of 5 amino acids can have the same mass by simple rearrangement of its constitutive amino acids; e.g., peptide VAGSE has the same mass as AVGSE or AEVGS and so on. For this technique to be successful, the masses of a large number of peptides must be obtained to provide enough specificity in the search, and this is not always possible. Mass redundancy occurs with greater frequency in large genomes. Moreover, peptide mass fingerprinting is effective only in the analysis of proteins from organisms whose genome is small, completely sequenced, and well annotated (131). It has limited use against unannotated or untranslated DNA databases such as the human genome. Because mass fingerprinting is not error tolerant, several factors in addition to mass redundancy contribute to its limited use, including sequencing errors, conservative substitutions, polymorphisms, and six possible translations at the DNA level.

Another factor affecting the success of peptide mass fingerprinting is mass accuracy (32, 62). Because it is critical to obtain an accurate measurement of the masses of multiple peptides, factors that alter the masses of those peptides can reduce the success of the method. One such example is the posttranslational modification of proteins. If the unknown protein is extensively modified, the peptides produced from that protein will not match the unmodified protein in the database. Recent improvements in the mass accuracy of mass spectrometers has increased the success rate of protein identification by this method (32, 54).

Finally, peptide mass fingerprinting does not work well with protein mixtures. As a protein mixture is converted to a mixture of peptides, it increases the complexity of the peptide mass fingerprint. The process of protein identification can be hindered if even two or three proteins are present in the sample (107). Several search methods have emerged to accommodate peptide mixtures in the mass spectrum. One example is a program called ProFound, which enables protein identification in simple protein mixtures (176). However, the lack of ability to analyze protein mixtures remains a major limitation of this method. A variety of tools for database searching now exist on the World Wide Web (Table 1). The ExPASy server provides a variety of tools for proteomics and programs for protein identification (reviewed in reference 165). Search programs used for peptide mass fingerprinting include PepSea (102), PeptIdent/MultiIdent (165), MS-Fit (32), MOWSE (125), and ProFound (176).

Amino acid sequence database searching. The most specific type of database searching for protein identification uses peptide amino acid sequence. If the amino acid sequence of a peptide can be identified, it can be used to search databases to find the protein from which it was derived. One method which utilizes this information is peptide mass tag searching. In this method, a partial amino acid sequence is obtained by interpretation of the MS/MS spectrum (the sequence tag) and this information is combined with the mass of the peptide and the masses of the peptide on either side of the sequence tag where the sequence is not known (Fig. 10). Also included in the search is the type of protease used to produce the peptides. Peptide mass tag searching is a more specific tool for protein identification than peptide mass fingerprinting (49, 103, 115, 170). In addition, one of the biggest advantages of utilizing MS/MS to obtain peptide amino acid sequence is that, unlike peptide mass fingerprinting, it is compatible with protein mixtures. The ability to identify proteins in mixtures is one of the great advantages of using MS as a protein identification tool. For example, in our laboratory we frequently identify multiple proteins from what appears to be a single band on an SDS-gel. In fact, in the majority of proteomics experiments, proteins are present in mixtures at the time of analysis.

View larger version (13K): [in this window] [in a new window]   FIG. 10. Peptide mass tag searching. Shown is a schematic of how information from an unknown peptide (top) is matched to a peptide sequence in a database (bottom) for protein identification. The partial amino acid sequence or "tag" obtained by MS/MS is combined with the peptide mass (parent mass), the mass of the peptide at the start of the sequence (mass tag 1), and the mass of the peptide at the end of the sequence (mass tag 2). The specificity of the protease used (trypsin is shown) can also be included in the search (103).

 
The major disadvantage of performing MS/MS is that the process is not easily automated. As a result, considerable time is expended in performing the analysis and interpreting the mass spectrum. Although computer programs can assist in the interpretation of the spectrum, they currently are not able to make accurate assignments without some guidance. In addition, when searching a database with peptide mass tags, there is a lack of flexibility in the search programs. If a single mistake is made in the assignment of a y- or b-ion (which can happen quite frequently), the amino acid sequence will be incorrect and the database search will bring up irrelevant proteins. Often it is necessary to confirm that the peptide sequence obtained from the database matches the sequence obtained in the mass spectrometer. This can be done by performing a theoretical fragmentation of the peptide from the database and comparing the two mass spectra. Additional clues can also be used, such as verifying if the peptide obtained from the database ends in amino acids consistent with the type of protease used.

De novo peptide sequence information. Another approach to protein identification is to obtain de novo sequence data from peptides by MS/MS and then use all the peptide sequences to search appropriate databases. Multiple peptide sequences can be used for protein identification by searching databases with the FASTS program (Mackey et al., submitted) (Fig. 5). The single biggest advantage of this method is the capability of searching peptide sequence information across both DNA and protein databases. This is because the search engine utilized exhibits a certain amount of flexibility in the assignment of protein scores. This search method is useful for organisms that do not have well-annotated databases such as Xenopus laevis or human. However, because this method requires several peptide amino acid sequences of 3 or 4 amino acids, it is not the first choice for peptide identification. Rather, the much faster methods of peptide mass fingerprinting or peptide mass tag searching can be used first. If these search methods fail, de novo sequence information can be obtained and used to identify the protein.

Uninterpreted MS/MS data searching. A large number of programs are now available for the identification of proteins by using uninterpreted MS/MS data. Examples include programs such as Mascot (129), SONAR (53), and SEQUEST (49) (Table 1). However, searches against unannotated or untranslated DNA databases with uninterpreted MS/MS data are likely to suffer from the same pitfalls associated with mass fingerprinting. In particular, polymorphisms, sequencing errors, and conservative substitutions will probably contribute to failure to accurately identify a protein. The development of uninterpreted MS/MS search algorithms that are error tolerant may overcome some of these shortcomings, provided that they assign some form of statistical scoring to the identified proteins.

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