Ultrasensitive Microscale Proteomic Analysis of Limited Availability Clinical Samples Using Porous Layer Open Tubular (PLOT) Liquid Chromatography Mass Spectrometry.

摘要

The limited availability and heterogeneity of human tissue for diagnostics and clinical research is a general constraint that spans many diseases, most notably cancer. Needle biopsy samples from tissue may contain only low thousands of cells, and be heterogeneous, e.g. tumors. Specific cell types, for example, stem cells, may be isolated from bone marrow, but their number is extremely low. While genomic analysis can be conducted for such small numbers of cells using DNA or RNA amplification, high sensitivity proteomic technologies still need to be developed to attack the difficult problem of deep analysis of clinical samples where the amount of sample is severely limited. There are several aspects of analysis at low sample amount including (1) sample procurement, (2) sample preparation, (3) LC, (4) MS, (5) data processing, and (6) bioinformatic analysis. Here we focus on LC and MS. We have developed porous layer open tubular (PLOT) columns for low level ultratrace analysis. We use 10 μm i.d. tube diameters and coat a layer of 1 to 2 μm of styrene-divinylbenzene (PS-DVB) on the inner wall. Flow rates of 20 nL/min are used and the MS is either an LTQ-Orbitrap XL or Q-Exactive (Thermo Fisher Scientific).