ABSTRACT
The preparation of complex biological samples for high-throughput mass spectrometric analyses remains a significant bottleneck, limiting advancement of the capabilities of mass spectrometry (MS) and ultimately limiting development of novel clinical assays. The removal of interfering species (e.g., salts, detergents, and buffers), concentration of dilute analytes, and the reduction of sample complexity are required in order to maximize the quality of resultant MS data. This study describes a novel sample preparation method that makes use of electrophoresis to prepare complex biological samples for high-throughput MS analysis. The method provides for integration of key sample preparation steps, including depletion, fractionation, desalting, and concentration. The prepared samples are captured onto a monolithic reversed-phase capture target that can be analyzed directly by a mass spectrometer. Up to 96 individual samples are simultaneously prepared for MS analysis in under 1 h. For standard proteins added to serum, this method provides femtomole level sensitivity and reproducible label-free detection (coefficient of variation <30%). This study demonstrates that this electrophoretic sample preparation system permits high-throughput sample preparation for mass spectrometric analysis of complex biological samples, such as serum, plasma, and tissue extracts.
Subject(s)
Electrophoresis/methods , Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Blood Proteins/analysis , Corticotropin-Like Intermediate Lobe Peptide/analysis , Electrophoresis/instrumentation , Humans , Liver/chemistry , Mice , Molecular Weight , Proteome/analysis , Reproducibility of Results , Sensitivity and Specificity , Serum Albumin, Bovine/analysis , Tissue Extracts/analysisABSTRACT
A polymer microfabricated proteomic sample preparation and MALDI MS sample presentation device, the integrated selective enrichment target (ISET), comprising an array of perforated nanovials is reported. Each perforated nanovial can be filled with selective extraction media (microbeads) for purification and concentration of protein/peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The main areas covered are the influence of the molding-process-induced surface roughness and how to address the lack of inherent conductivity in the polyetheretherketone (PEEK) material for optimal MALDI MS readout. Application of the disposable polymeric ISET devices for solid-phase extraction and phosphopeptide capture is also demonstrated.
