A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from twodimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of twodimensional gel spots. The method was applied to a twodimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identiy.The identification of a purified protein is necessary in many areas of biochemical research. As the resolution and sensitivity of purification tools increase, the demand for protein sequencing increases. For example, a single high-resolution two-dimensional polyacrylamide gel can separate hundreds of proteins (1,2). Identification of all the resolvable proteins on a two-dimensional gel by conventional protein sequencing is a daunting task. The correlation of DNA from large-scale sequencing projects with their protein products will continue to place increasing demands upon protein sequencing.Proteins that are N-terminally blocked present an additional challenge since they cannot be directly sequenced by Edman degradation. Blockage may occur by posttranslational modification during protein synthesis or during purification. Many intracellular proteins have been reported to be N-terminally acetylated (3). In order to obtain internal sequence on a blocked protein, 50-100 pmol of material is usually required. The blocked protein is chemically or enzymatically cleaved. The peptides are then separated by HPLC and sequenced, a process which can take 3-4 days. In addition, proteins initially thought to be novel may, after purification and sequencing, be found already to exist in the protein sequence database. As a result, a significant fraction of sequencer time is spent simply identifying known proteins.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 1...

-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically recognize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase by purified p24. p24 is found only in retina, and it copurifies with outer segment membranes. Immunocytochemical analysis shows that it is present in rod photoreceptor cells. An immobilized antibody column was used to purify p24 from a heat-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogenous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca 2؉ inhibits this activation with an EC 50 near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca 2؉ sensitivity to photoreceptor membrane guanylyl cyclase. We propose that p24 be referred to as GCAP-2 and that GCAP be referred to as GCAP-1.

The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.

Peptide mass fingerprinting (PMF) grew from a need for a faster, more efficient method to identify frequently observed proteins in electrophoresis gels. We describe the genesis of the idea in 1989, and show the first demonstration with fast atom bombardment mass spectrometry. Despite its promise, the method was seldom used until 1992, with the coming of significantly more sensitive commercial instrumentation based on MALDI-TOF-MS. We recount the evolution of the method and its dependence on a number of technical breakthroughs, both in mass spectrometry and in other areas. We show how it laid the foundation for high-throughput, high-sensitivity methods of protein analysis, now known as proteomics. We conclude with recommendations for further improvements, and speculation of the role of PMF in the future. (J Am Soc Mass Spectrom 2003, 14, 931-942)

A method is described for rapid purification of synthetic oligodeoxynucleotides to remove sodium counter ions prior to electrospray ionization mass spectrometry. The oligomers, following purification by gel electrophoresis, are precipitated from ammonium acetate to replace sodium ions by ammonium ions, and dissolved in water. Negative-ion electrospray spectra are presented for oligomers with up to 48 residues in which the most intense peaks correspond to the [M -nHY-ion. The spectrum of 77-mer (M, 24 039) shows the predominant peak as due to retention of only a single sodium ion. Changes in the spectra are reported for different instrument orifice voltages and different solution pH. The method should permit rapid, accurate analysis of most typical, synthetic oligodeoxynucleotides.

A number of proteins from a silver-stained two-dimensional (2-D) electrophoresis gel of mouse liver whole-cell lysate were identified by peptide mass mapping and sequence database searching. The excised protein spots were processed by in situ reduction and alkylation, followed by Lys-C digestion. The masses of the resulting peptide mixtures were measured with a matrix-assisted laser desorption/ionization (MALDI) reflection-time-of-flight mass spectrometer. These masses were used successfully to search a protein sequence database. Optimized silver staining and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provided by delayed extraction were important for high specificity in the database search. Fragment ion data obtained by MALDI post-source decay (PSD) measurements not only provided confirmation of peptide identification, but could be used to identify the protein from a single peptide without spectral interpretation.

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymatic digestion of phosphoroteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combmation allows separation and mass spectrometric identification of phosphorylated and nonphosphorylated peptides. In this study, the feasibility and general applicability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides. The sensitivity and practicality of the technique for phosphoproteins are demonstrated via the analysis of 30 pmol (∼0.7 μg) of bovine β-casein purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and digested in situ with trypsin. It is observed that on-line IMAC/ESI/MS suffers less from sample losses than experiments performed off-line, suggesting that the limiting factors in sensitivity for this technique are the purification procedures and sample handling rather than the IMAC and mass spectrometry. Thus, the ability to inject the tryptic digest of an electroblotted protein directly onto the column without buffer exchange and to analyze the eluent directly via on-line coupling of the IMAC column to the mass spectrometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.

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