Abstract
Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.
Highlights
Proteins are essential cellular machinery, performing and enabling tasks within biological systems.The variety of proteins is extensive, and the role they occupy in biology is deep and complex; life depends on proteins
The weakness of Capillary zone electrophoresis (CZE) is its low loading amount, due in part to the small volume of the 50 μm inner diameter (ID) capillary and zero peptide retention on the stationary phase. This analysis shows that CZE is a viable micro- to nano-proteomic separation technique, lowering sample requirements while retaining sensitivity and providing numerous peptide and protein identifications, albeit complementary to ultra-performance liquid chromatography (UPLC)
Two-dimensional (2D) cell culture and murine model are both useful model systems with unique advantages and disadvantages. 2D cell culture is least likely to be used for microproteomic analysis; great quantities of cells can be cultured in a single flask, resulting in milligram amounts of protein available
Summary
Proteins are essential cellular machinery, performing and enabling tasks within biological systems. The field of proteomics is the large-scale study of proteins and the proteome and encompasses many techniques, such as immunoassays [1] and two-dimensional differential gel electrophoresis (2-D DIGE) [2,3] Another group of methodologies that are growing in popularity for protein discovery and analyses are mass spectrometry-based approaches. There are two main approaches for mass spectrometry-based proteomics, top–down and bottom–up analyses. By pairing mass analyzers and detectors, adding equipment in different configurations, and coupling separations and mass spectrometers together, there are virtually limitless possibilities, functionalities, and speeds of data acquisition for mass spectrometry-based proteomic analysis. This review will focus on sample preparation strategies for bottom–up mass spectrometry-based proteomics, with brief focus on obtaining samples for analysis
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