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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 ...

Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate.

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A characteristic feature of cellular signal transduction pathways in eukaryotes is the separation of catalysis from target recognition. Several modular domains that recognize short peptide sequences and target signaling proteins to these sequences have been identified. The structural bases of the specificities of recognition by SH2, SH3, and PTB domains have been elucidated by X-ray crystallography and NMR, and these results are reviewed here. In addition, the mechanism of cooperative interactions between these domains is discussed.