Mass spectrometry (MS) based proteomic technologies are currently in the forefront for the discovery of blood borne biomarkers but despite the recent technological advances, analytical and physiological barriers hinder the biological marker discovery: the reality is that analytes that likely represent the most clinical important markers are too low in abundance for even the most sophisticated MS technology to identify. Recent interest has focused on the low molecular weight (LMW) \#8220;peptidome\#8221; as a potentially rich and relatively unexplored source of circulating markers. These analytes may be small proteins or fragment isoforms of proteins that are produced by and within the unique tumor microenvironment. In this work we have originated a novel workflow for body fluid-based biomarker discovery, in which hydrogel nanoparticles are engineered to amplify, fractionate and enrich LMW analytes at once, in just a few minutes from raw unprocessed serum, providing a powerful pre-analytical method for mass spectrometry-based biomarker discovery. Hydrogel nanoparticles consist of a N-isopropylacrylamide (NIPAm) core, conjugated to an affinity based bait, and surrounded by a NIPAm shell. LMW proteins and peptides are attracted by the bait into the particles while the shell acts as a sieve, excluding high molecular weight, high abundance proteins such as albumin. Proteins eluted from the particles are analyzed using a Thermo LTQ-Orbitrap mass spectrometer. The MS data are searched against a human protein database (NCBI) with SEQUEST Spectral counting (MS2-based) analysis is accomplished using Scaffold (Proteome Software, Inc.). The MS1-based comparative analysis is accomplished using the SIEVE software (Thermo and Vast Scientific). Candidate differentially abundant peptides are evaluated by manual inspection of the raw data to confirm peptide identification and determine intensities which would provide us with statistics conveying the differential expression of the protein biomarkers. This protocol is being applied to serum from patients with prostate cancer whereby serum was collected pre and post prostatectomy in order to select biomarkers for the early diagnosis of clinical relevant prostate cancer.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4487.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO