Funds are requested for a ionspray triple-quadrupole mass spectrometer for use in biological research involving proteins. The instrument requested a wide range of protein samples in solution, in total amounts of a few to a few tens of picomoles per experiment (approx. 1mugram; 20 picomoles of a 25kDa protein is 0.5mugram). Information generated includes the precise mass of the intact protein or components of a protein mixture (mass accuracy; approx.+/-3dalton for a 25kDa protein). Individual peptides, even in the complex mixtures present in the chemical or enzymatic digest of a few tens of picomoles of a protein, can be studies directly or after prior separation of the peptides by reverse phase HPLC. For peptides up to 20 or 30 amino acids in length, mass accuracy of better that 1 dalton is obtained. Such precise mass information is a general method for detecting and defining post-translational modifications, inc. disulfide bond locations, of proteins. The data also serve as a precise way of correlating a gene with its translation product, revealing not only covalent modifications but also N- or C-terminal """"""""clipping"""""""" of the protein. An additional capability of the instrument described is mass spectrometry- mass spectrometry experiments, using Collison-Induced-Dissociation to generate sequence-defining fragments from a single component of a mixture of peptides or proteins. Frequently, such mass spectra are too complex for complete interpretation, but the data can be enormously useful in defining the exact nature and site of post-translational modifications, such as the exact site and nature of phosphorylation of peptide. Carbohydrate moieties on glycopeptides can also yield structure-defining fragmentation patterns. More rarely, MS/MS can generate limited amino acid sequence data from blocked proteins, especially from tryptic digests which can give interpretable fragmentation patterns in up to 20: or so of peptides present in a digest. This can be useful in generating oligonucleotides for isolation f the corresponding gene. The capabilities described have important uses in a wide range of biological research. The instrument will have a profound impact on the effectiveness of a number of NIH-sponsored programs.
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