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.

Understanding cancer metastasis at the proteoform level is crucial for discovering previously unknown protein biomarkers for cancer diagnosis and drug development. We present the first top-down proteomics (TDP) study of a pair of isogenic human nonmetastatic and metastatic colorectal cancer (CRC) cell lines (SW480 and SW620). We identified 23,622 proteoforms of 2332 proteins from the two cell lines, representing nearly fivefold improvement in the number of proteoform identifications (IDs) compared to previous TDP datasets of human cancer cells. We revealed substantial differences between the SW480 and SW620 cell lines regarding proteoform and single amino acid variant (SAAV) profiles. Quantitative TDP unveiled differentially expressed proteoforms between the two cell lines, and the corresponding genes had diversified functions and were closely related to cancer. Our study represents a pivotal advance in TDP toward the characterization of human proteome in a proteoform-specific manner, which will transform basic and translational biomedical research.

Sepsis is a serious medical condition that occurs in 30% of patients in intensive care units (ICUs). Early detection of sepsis is key to prevent its progression to severe sepsis and septic shock, which can cause organ failure and death. Diagnostic criteria for sepsis are nonspecific and hinder a timely diagnosis in patients. Therefore, there is currently a large effort to detect biomarkers that can aid physicians in the diagnosis and prognosis of sepsis. Mass spectrometry is often the method of choice to detect metabolomic and proteomic changes that occur during sepsis progression. These “omics” strategies allow for untargeted profiling of thousands of metabolites and proteins from human biological samples obtained from septic patients. Differential expression of or modifications to these metabolites and proteins can provide a more reliable source of diagnostic biomarkers for sepsis. Here, we focus on the current knowledge of biomarkers of sepsis and discuss the various mass spectrometric technologies used in their detection. We consider studies of the metabolome and proteome and summarize information regarding potential biomarkers in both general and neonatal sepsis.