Analysis Of Peptides, Proteins, And Metabolites Using MS

Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

This year we are again participating in the many projects relating to the analysis of peptides and proteins that have been brought to our attention by researchers at NHLBI and NIH. The goal is still to use advanced mass spectrometric technology to study the biology of a particular system by obtaining information on the identity of proteins either through mass spectrometric sequencing or by simply carefully measuring the masses of a sufficient number of its peptides. This technique is unique in identifying post-translational modifications (PTMs) essential for protein/cellular functions. With the state-of-the-art Micromass QTOF3 mass spectrometer and other LC-MS and MALDI-TOF instruments, we were able to perform many experiments on biological samples, mostly in this protein and peptide class. In addition,we seek new chemical methods to enhance the search routines in order to increase their reliability. Studies have been initiated on the use of nitrites as derivitising reagents for peptide analysis. We have published on the identification of all of the sites of glycosylation on rat zona pellucida as well as its N- and C- terminal amino acids along with its disulfide linkages. We are still studying a compound with activity in reducing multidrug resistance in chemotherapy and have prepared a radioactive form (J. Ludwig, G. Szakacs, M. Gottesman)and the drug is soon to go into preclinical trial. We have examined the structures of proteins whose cysteine groups react with peroxide and identified the location of oxidation (S.G. Rhee, NHLBI). We continue to spend a significant amount of time optimizing sensitivity and resolution of our QTOF3,its capillary LC and its communication with the mass spectrometer, in particular with regard to its ability to analyze proteins as opposed to peptides. The APCI method is still being reexamined for its potential in analysis of peptides. The 2D-LC ProteomeX system from ThermoFinnigan is still under investigation as a source of high throughput analysis but it has been frought with several problems in regard to its chromatographic system.