Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

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.

Development of an Analytical Method for the Metaproteomic Investigation of Bioaerosol from Work Environments

“…In fact, sample losses become crippling with precipitation procedures from mass‐limited samples, as they make it challenging to obtain sufficient amounts of proteins to generate high‐quality MS data. To overcome this challenge, a microscale technique, that is, filter aided sample preparation (FASP), was applied to protocols A, B, and C, as it is particularly suited for low sample sizes (100 µg or lower) and still enables an excellent proteome coverage . We tested two FASP devices with different molecular weight cutoffs (10 and 30 kDa); however, both clogged due to the presence of “carbonaceous aerosols” that remain in solution after centrifugation; therefore, no data were obtained by the FASP method variations and no further modifications were tested on protocols A, B, and C.…”

Can LC and LC-MS ever replace immunoassays?

“…Hence, in most LC-MS experiments, the proteins of interest are enzymatically digested, typically with trypsin, to produce smaller peptides to aid chromatographic separation prior to mass spectrometry and additionally the mass spectrometer is more sensitive to peptides than proteins. For a recent review of sample preparation for protein analysis in LC-MS see Feist and Hummon [12]. The sensitivity for a specific peptide, natural or derived from a protein by enzymatic digestion with an enzyme such as trypsin, or protein will vary.…”

Recent developments and applications of capillary and microchip electrophoresis in proteomic and peptidomic analyses

“…The proteins in biological samples (e.g. biopsies, tissue extracts, body fluids) must be made accessible by lysis and extraction from the cells . Frequently detergent lysis (e.g.…”

“…by Triton X‐100, Nonidet P‐40, Tween 20, Tween 80, octyl glycoside etc.) in combination with some mechanical stimulus that physically breaks the cells is applied . Once isolated, proteins in their native state are often insoluble.…”

“…In bottom‐up proteomics, the protein samples are first digested with enzymes to generate peptides, followed by separation (usually RPLC or CE) and MS/MS detection. The obtained MS/MS spectra are then analyzed through correlation against database .…”