Microproteomics: Quantitative Proteomic Profiling of Small Numbers of Laser-Captured Cells

Abstract

INTRODUCTIONDuring the last decade, significant progress in the analysis of whole genomes and transcriptomes has triggered efforts to analyze the proteome. Advancements in protein extraction, purification, and identification have been driven by the development of mass spectrometers with greater sensitivity and resolution. Nevertheless, comparative and quantitative proteomic technologies have not progressed to the extent of genomic and transcriptomic technologies for accessing gene expression differences. Unlike the genome, which is similar throughout all cells in a given organism, the proteome varies in different cells. Also, there is no self-replicating amplification mechanism for proteins such as the polymerase chain reaction (PCR) for DNA. Therefore, developing methods that extract, separate, detect, and identify proteins from extremely small samples are needed. The advent of laser capture microdissection (LCM) has expanded the analytical capabilities of proteomics. LCM has proven an effective technique to harvest pure cell populations from tissue sections. This protocol describes a microproteomic platform that uses nanoscale liquid chromatography/tandem mass spectrometry (nano-LC-MS/MS) to simultaneously identify and quantify hundreds of proteins from LCMs of tissue sections from small tissue samples containing as few as 1000 cells. The LCM-dissected tissues are subjected to protein extraction, reduction, alkylation, and digestion, followed by injection into a nano-LC-MS/MS system for chromatographic separation and protein identification. The approach can be validated by secondary screening using immunological techniques such as immunohistochemistry or immunoblots.

STAT3‐dependent analysis revealsPDK4as independent predictor of recurrence in prostate cancer

“…Finally, purified peptides were resolved in 2% acetonitrile and 0.1% TFA, and the entire sample was injected for MS analysis in a single‐shot measurement. Protocols were adapted from Roulhac et al () and Wang et al ().…”

Proteomic analysis of dentin–enamel junction and adjacent protein‐containing enamel matrix layer of healthy human molar teeth

“…The second extraction method (Method 2) was adapted from a published protocol . Briefly, 200 μ l of a mixture of 50 mM ammonium bicarbonate with TFE (1:1, v/v) was added to a vial containing an LCM sample to initiate protein extraction.…”

“…The LCM system represents a modified microscopic technique and allows selection of cells within histological sections and their contact‐free laser‐mediated catapult into a test tube. The LCM‐dissected tissues are subjected to protein extraction, reduction, alkylation, and digestion, followed by injection into a nano‐liquid chromatography coupled to tandem mass spectrometry (nLC‐MS/MS) system for chromatographic analysis and protein identification .…”

Convergent transcriptional specializations in the brains of humans and song-learning birds

“…To determine whether the gene expression specializations we found in RA were reflected in the corresponding protein products, we performed nanoscale capillary tandem mass spectrometry experiments on zebra finch RA and the adjacent mAC with a microproteomic method developed for laser capture–microdissected cells (74). We identified 81 proteins as differentially expressed in RA (fig.…”