PNNL

Abstract

Human degradome and its peptidome products are closely related with many pathologic states including cancer. Detection of the peptidome products can provide information of the protein degradation and the activity of the intercellular and intracellular proteases related to diseases. In this work, we describe the AC/SEC-HRLC-FT MS/MS-UStags strategy for top-down analysis of the human blood peptidome components and their modifications. The human blood peptidome is isolated through application of AC/SEC, which enriches its components by >300-fold. These components are separated by the long column HRLC providing a peak capacity of ~300 for the components having MW of up to 20 kDa under the condition of elongated elution. The separated species are identified by the FT MS/MS-UStags sequencing method. From the examined blood plasma sampled from a healthy person, we identified >200 peptidome peptides that originated from 29 degradome substrates from the IPI human protein database (~70,000 entries) without the identification errors. The peptidome peptide sequence mutations and modifications, including acetylation, cysteinylation, acetylhexosamine, oxidation, didehydro, amidation, and pyro-glu, were observed for the peptidome components. Intact low molecular weight proteins were observed only having modified forms. The strategy described here is now used to study the degradation activities in the early-stage breast cancer patient blood, including the selectivity for degradome substrates and functional domains, the peptide cleavage specificity, and the magnitude of degradome substrate involvements in degradation, which provides the insights and footprints for the disease-related degradations in the blood.