MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

Proteolysis is an important but perhaps the most overlooked eukaryotic post-translational modification. The biology of neuropeptides [1], peptide hormones [2,3], and unusual proteolytically derived signaling molecules [4,5] stimulates interest in the establishment of appropriate analytical workflows. Mass spectrometry is one of the most useful methods for the analysis of complex peptide mixtures. Proteomic assays typically utilize sequence-specific proteases to characterize the components of complex protein mixtures [6] but the methods for analysis of naturally occurring peptides, without a proteolytic step, are less developed. Applications of mass spectrometry to the study of peptides in various body fluids including cerebrospinal fluid [7], urine [8], synovial fluid [9], saliva [10] and of course serum and plasma [11,12] have been described. A universally useful method for the preparation of the peptides for analysis has not yet emerged and context-specific optimization is typically required. The reported methods include ultrafiltration [13], precipitation by organic solvents [14], solid phase extraction [15], size-exclusion chromatography [11], differential solubilization method [16], and nanoparticle trapping technology [17]. Even methods as simple as direct MALDI-TOF analysis of a complex mixture in a body fluids were used successfully [12] with the benefit of high-throughput, minimal preparative losses of analytes, and minimal sample requirements. Indeed, MALDI- or SELDI-TOF based analyses are most likely the richest source of information about native peptides. On the other hand, these methods suffer from inherent quantitative limitations [15,18]. The original semi quantitative screens are therefore followed by the development of isotope dilution kinetic assays [19] and, most recently, multiple reaction monitoring (MRM) LC-MS/MS quantification of target peptides [20].MRM has emerged as an LC-MS alternative to antibody based assays for accurate protein quantifi