Protein concentration is one of the most basic and important parameters in functional analysis because the kinetics/dynamics of cellular molecules is described by changes in the concentrations of proteins in particular compartments. Mass spectrometry (MS) plays a central role in proteome studies and allows protein identification and quantitation at the same time. An MS-based method allows simultaneous identification and quantitation of individual proteins and determination of changes in the levels of modifications at specific sites on individual proteins. Accurate quantitation is achieved through the use of whole cell stable isotope labeling, and the seminal work on differential metabolic labeling of proteins by Oda et al. has led to many strategies for quantitative proteomics including quantitative tissue proteome analysis based on culture-derived isotope tag (CDIT). This is a generalized approach and affords a quantitative description of cellular differences at the level of protein expression and modification, thus providing information that is critical to the understanding of complex biological phenomena. In addition to this relative quantitative proteomics, MS-based absolute quantitation is also possible by adding a known amount of standard peptides (e.g. isotope dilution), or reverse isotope dilution (CDIT).
Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry, it can be difficult to elucidate protein kinase substrates in the presence of a large excess of relatively abundant non-phosphoproteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, we developed a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts after chemical replacement of the phosphate moieties by affinity tags. Although this is an interesting method, the procedure is complicated and of low efficiency. Therefore, we developed a simple, highly specific enrichment procedure for phosphopeptides, by increasing the specificity of an immobilized metal affinity column (IMAC) without using any chemical reaction. The increase in selectivity was achieved by (a) using a strong acid to discriminate phosphates from carboxyl groups of peptides, (b) using a high concentration of acetonitrile to remove hydrophobic non-phosphopeptides, and (c) desalting the phosphate buffer used for competitive elution from the IMAC. By applying this efficient approach, we were able to identify more than one thousand phosphopeptides.
Chemical proteomics is an effective approach to focused proteomics, having the potential to find specific interactors in moderate-scale comprehensive analysis. Unlike chemical genetics, chemical proteomics directly and comprehensively identifies proteins that bind specifically to candidate compounds by means of affinity chromatographic purification using the immobilized candidate, combined with mass spectrometric identification of interacting proteins. This is an effective approach for discovering unknown protein functions, identifying the molecular mechanisms of drug action, and obtaining information for optimization of lead compounds. However, immobilized-small molecule affinity chromatography always suffers from the problem of nonspecific binders. Although several approaches have been reported to reduce nonspecific binding proteins, these are mainly focused on the use of low-binding-affinity beads or insertion of a spacer between the bead and the compound. Stable isotope labeling strategies have proven particularly advantageous for the discrimination of true interactors from many nonspecific binders, including carrier proteins such as serum albumin, and are expected to be valuable for drug discovery.

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We investigated the electron capture dissociation (ECD) of triantennary complex-type N-glycosylated peptides prepared from bovine fetuin. Previous reports on Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) suggested that ECD is an advantageous method for structural characterization of glycopeptides, because it can cleave the N-Cα bond on the peptide backbone while retaining the labile glycosidic bonds. We present here new data from ECD which resulted in the degradation of the glycan structure prior to any backbone cleavage. Based on the ECD experiment on deglycosylated samples, the character of the peptide backbone sequence, which contains carbamoylmethylated cysteine residue, seemed to prevent extensive N-Cα bond cleavage. It appears that more basic investigations are necessary to promote practical use of ECD for structural characterization of complex glycopeptides because each type of glycopeptide exhibits particular fragmentation patterns.

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Metabolite identification is a critical step in drug discovery and development. Finding metabolites by comparing sample data with control data is both difficult and tedious, despite the various software applications available. The objective of this work was to detect metabolites from liquid chromatography coupled mass spectrometry/mass spectrometry (MS/MS) data by using only the product ion mass spectrum. Molecules that have similar structures can be selected easily and rapidly from auto MS/MS data using partial least squares (PLS) for the product ion mass spectra. Major metabolites of verapamil from the fraction in the presence of Phase I co-factors are well known. Automated ion trap MS/MS data-dependent acquisition with PLS was successfully applied and the major known metabolites and one unexpected metabolite were detected. Their structures are elucidated in this paper. It is suggested that automated ion trap MS/MS data-dependent acquisition with PLS can be a useful tool in detecting metabolites and elucidating their structure.

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To detect hidden explosives, we have developed an explosives trace detector based on atmospheric pressure chemical ionization mass spectrometry (APCI-MS). In our APCI ion source, the direction of the sample gas flow introduced into the ion source is opposite to that of the ion flow produced by the ion source. This novel ion source has several advantages: for example, high ionization efficiency for nitro-compounds, and a long lifetime without maintenance. Our explosives trace detector model DS-110E has received certification from the Transportation Security Administration (TSA) in 1995.

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Ions are not organisms, and thus they do not proliferate by either sexual or asexual reproduction. They are hence incapable of being parents, children, or grandchildren. Therefore, the terms “parent ion,” “daughter ion,” and “granddaughter ion” are scientifically incorrect. In addition, mass spectrometrists should note that the terms “daughter ion” and “granddaughter ion” are gender-specific. (The terms “daughter nuclide” and “granddaughter nuclide” are also gender-specific.)
Therefore, the terms “parent ion” and “daughter ion” need to be replaced by “precursor ion” and “product ion,” respectively. Instead of the gender-specific terms “granddaughter ion” and “great-granddaughter ion,” the gender-neutral terms “second generation product ion” and “third generation product ion” should be used.

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