Proteomic LC-MS approaches combined with genome-annotated databases currently allow identification of thousands of proteins from complex mixtures (1). Approaches have also been developed for relative quantitation using stable isotope labeling (2-4). Recently not only comprehensive quantitation studies between two states (5, 6) but also protein-protein (7, 8), protein-peptide (9), and protein-drug (10) interaction analyses have been reported. So far, however, a comprehensive approach for determining protein concentrations in one sample has not been established. Protein concentrations are one of the most basic and important parameters in quantitative proteomics because the kinetics/dynamics of the cellular proteome is described in terms of changes in the concentrations of proteins in particular compartments. Biological experiments often require at least some information on protein abundance for correct interpretation. In the past, crude quantitative information could be drawn from the intensity of gel staining in comparison to a known amount of marker protein. However, in complex mixture analysis, individual proteins cannot be stained individually, and usually all information about protein abundance is lost. So far, isotope-labeled synthetic peptides have been used as internal standards for absolute quantitation of particular proteins of interest (11,12). This approach is in principle applicable to comprehensive analysis but is hampered by the high cost of isotope-labeled peptides as well as the difficulty of quantitative digestion of proteins in-gel (13).Even a single nano-LC-MS/MS analysis can easily generate a long list of identified proteins with the help of database searching, and additional information can be extracted, such as the hit rank in identification, the probability score, the number of identified peptides per protein, ion counts of identified peptides, LC retention times, and so on. Qualitatively some parameters, such as the hit rank, the score, and the number of peptides per protein (14), can be considered as indicators for protein abundance in the analyzed sample. Among them, the integrated ion counts of the peptides identifying each protein would be the most direct parameter to describe the abundance and has been used to compare protein expression in different states (15). However, a mass spectrometer is not as versatile as an absorbance detector because of limited linearity and possibly because of background and ionization suppression effects (16). Therefore, it is necessary to normalize these parameters to obtain at least approximate quantitative information. The first approach to achieve this, to our knowledge, was to use the number of peptides per protein normalized by the theoretical number of

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron-ionization mass spectrometry (EI-MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)-MS(n) data of 2337 authentic compounds of metabolites, 11 545 EI-MS and 834 other-MS data of 10,286 volatile natural and synthetic compounds, and 3045 ESI-MS(2) data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI-MS(2) data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass-to-charge ratio are optimized to the ESI-MS(2) data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI-MS(2) data on an identical compound under different collision-induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21-23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data.

A mass spectrometry-based method is described for simultaneous identification and quantitation of individual proteins and for determining changes in the levels of modifications at specific sites on individual proteins. Accurate quantitation is achieved through the use of wholecell stable isotope labeling. This approach was applied to the detection of abundance differences of proteins present in wild-type versus mutant cell populations and to the identification of in vivo phosphorylation sites in the PAK-related yeast Ste20 protein kinase that depend specifically on the G1 cyclin Cln2. The present method is general 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.The ongoing accumulation of vast collections of DNA sequence data has catalyzed the development of novel approaches for profiling the expression of genes at the mRNA level. These methods, while extraordinarily powerful, do not provide direct information on changes, either in the levels of proteins or their states of modification. The development of analogous high throughput methods for directly monitoring protein levels, while increasingly desirable for biological investigations in the postgenome era (1-3), presents a formidable analytical challenge. Although recent advances in the use of mass spectrometry (MS) in conjunction with protein͞DNA-sequence database search-algorithms allow for the identification of proteins with unprecedented speed (4-7), it remains difficult to obtain accurate quantitative information concerning the levels of the identified proteins and the levels of site-specific modifications within individual protein molecules. In the absence of appropriate antibodies, quantitation is usually achieved by autoradiography after metabolic radiolabeling, fluorography, or the use of protein stains. These procedures depend on complete separation of the proteins of interest by techniques such as high-resolution two-dimensional electrophoresis (8, 9). There remains a pressing need for easier, more reliable means to rapidly profile protein levels. Here we describe a general method for accurately comparing levels of individual proteins present in cell pools that differ in some respect from one another (e.g., the presence of a mutated gene) and for accurately determining changes in the levels of modifications (e.g., phosphorylation) at specific sites on the individual proteins. The procedure can be applied to mixtures of proteins, obviating the need for complete separation. MATERIALS AND METHODSMatrix-Assisted Laser Desorption͞Ionization Mass Spectrometric (MALDI-MS) Tryptic Maps. MALDI-MS (10) tryptic maps of protein gel-bands were obtained as follows.Individual protein bands were excised, destained, washed, and digested with modified trypsin (Boehringer Mannheim), and the resulting peptides were extracted with acetonitrile. After vacuum drying, each sample was redissolved in 5 l ...

The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.