Technique development of high-throughput and high-sensitivity sample preparation and separation for proteomics

Sample preparation and separation methods determine the sensitivity and the quantification accuracy of the proteomics analysis. This article covers a comprehensive review of the recent technique development of high-throughput and high-sensitivity sample preparation and separation methods in proteomics research.

Differential Expression Study of Lysine Crotonylation and Proteome for Chronic Obstructive Pulmonary Disease Combined with Type II Respiratory Failure

Introduction. The modification of lysine crotonylation (Kcr) is another biological function of histone in addition to modification of lysine acetylation (Kac), which may play a specific regulatory role in diseases. Objectives. This study compared the expression levels of Kcr and proteome between patients with chronic obstructive pulmonary disease (COPD) combined with type II respiratory failure (RF) to study the relationship between Kcr, proteome, and COPD. Methods. We tested the Kcr and proteome of COPD combined with type II RF and normal control (NC) using croton acylation enrichment technology and liquid chromatography tandem mass spectrometry (LC-MS/MS) with high resolution. Results. We found that 32 sites of 23 proteins were upregulated and 914 sites of 295 proteins were downregulated. We performed Kyoto Encyclopedia of Genes and Genomes (KEGG), protein domain, and Gene Ontology (GO) analysis on crotonylated protein. In proteomics research, we found that 190 proteins were upregulated and 151 proteins were downregulated. Among them, 90 proteins were both modified by differentially expressed crotonylation sites and differentially expressed in COPD combined with type II RF and NC. Conclusion. Differentially expressed crotonylation sites may be involved in the development of COPD combined with type II RF. 90 proteins modified by crotonylation and differentially expressed in COPD combined with type II RF can be used as markers for the study of the molecular pathogenesis of COPD combined with type II RF.

Proteomics and ultrastructural analysis of Hermetia illucens (Diptera: Stratiomyidae) larval peritrophic matrix

Background The black soldier fly (Hermetia illucens) has significant economic potential. The larvae can be used in financially viable waste management systems, as they are voracious feeders able to efficiently convert low-quality waste into valuable biomass. However, most studies on H. illucens in recent decades have focused on optimizing their breeding and bioconversion conditions, while information on their biology is limited. Methods About 200 fifth instar well-fed larvae were sacrificed in this work. The liquid chromatography-tandem mass spectrometry and scanning electron microscopy were employed in this study to perform a proteomic and ultrastructural analysis of the peritrophic matrix (PM) of H. illucens larvae. Results A total of 565 proteins were identified in the PM samples of H. illucen, of which 177 proteins were predicted to contain signal peptides, bioinformatics analysis and manual curation determined 88 proteins may be associated with the PM, with functions in digestion, immunity, PM modulation, and others. The ultrastructure of the H. illucens larval PM observed by scanning electron microscopy shows a unique diamond-shaped chitin grid texture. Conclusions It is the first and most comprehensive proteomics research about the PM of H. illucens larvae to date. All the proteins identified in this work has been discussed in details, except several unnamed or uncharacterized proteins, which should not be ignored and need further study. A comparison of the ultrastructure between H. illucens larval PM and those of other insects as observed by SEM indicates that the PM displays diverse textures on an ultra-micro scale and we suscept a unique diamond-shaped chitin grid texture may help H. illucens larval to hold more food. This work deepens our understanding of the molecular architecture and ultrastructure of the H. illucens larval PM.

Bringing New Methods to the Seed Proteomics Platform: Challenges and Perspectives

For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins, and carbohydrates in their seeds, distinctly dominate modern agriculture, thus play an essential role in food industry and fuel production. Therefore, seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, seed development and germination as well as age- and stress-related alterations in seed vigor, longevity, nutritional value, and safety can be addressed by a broad panel of analytical, biochemical, and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes accompanying plant development, senescence, and response to biotic or abiotic stress. Among individual post-genomic methodological platforms, proteomics represents one of the most effective ones, giving access to cellular metabolism at the level of proteins. During the recent decades, multiple methodological advances were introduced in different branches of life science, although only some of them were established in seed proteomics so far. Therefore, here we discuss main methodological approaches already employed in seed proteomics, as well as those still waiting for implementation in this field of plant research, with a special emphasis on sample preparation, data acquisition, processing, and post-processing. Thereby, the overall goal of this review is to bring new methodologies emerging in different areas of proteomics research (clinical, food, ecological, microbial, and plant proteomics) to the broad society of seed biologists.

Identification and quantification of honeybee venom constituents by multiplatform metabolomics

Honeybee (Apis mellifera) venom (HBV) has been a subject of extensive proteomics research; however, scarce information on its metabolite composition can be found in the literature. The aim of the study was to identify and quantify the metabolites present in HBV. To gain the highest metabolite coverage, three different mass spectrometry (MS)-based methodologies were applied. In the first step, untargeted metabolomics was used, which employed high-resolution, accurate-mass Orbitrap MS. It allowed obtaining a broad overview of HBV metabolic components. Then, two targeted metabolomics approaches, which employed triple quadrupole MS, were applied to quantify metabolites in HBV samples. The untargeted metabolomics not only confirmed the presence of amines, amino acids, carbohydrates, and organic acids in HBV, but also provided information on venom components from other metabolite classes (e.g., nucleosides, alcohols, purine and pyrimidine derivatives). The combination of three MS-based metabolomics platforms facilitated the identification of 214 metabolites in HBV samples, among which 138 were quantified. The obtaining of the wide free amino acid profiles of HBV is one of the project’s achievements. Our study contributed significantly to broadening the knowledge about HBV composition and should be continued to obtain the most comprehensive metabolite profile of HBV.

Emerging mass spectrometry-based proteomics methodologies for novel biomedical applications

Research into the basic biology of human health and disease, as well as translational human research and clinical applications, all benefit from the growing accessibility and versatility of mass spectrometry (MS)-based proteomics. Although once limited in throughput and sensitivity, proteomic studies have quickly grown in scope and scale over the last decade due to significant advances in instrumentation, computational approaches, and bio-sample preparation. Here, we review these latest developments in MS and highlight how these techniques are used to study the mechanisms, diagnosis, and treatment of human diseases. We first describe recent groundbreaking technological advancements for MS-based proteomics, including novel data acquisition techniques and protein quantification approaches. Next, we describe innovations that enable the unprecedented depth of coverage in protein signaling and spatiotemporal protein distributions, including studies of post-translational modifications, protein turnover, and single-cell proteomics. Finally, we explore new workflows to investigate protein complexes and structures, and we present new approaches for protein–protein interaction studies and intact protein or top-down MS. While these approaches are only recently incipient, we anticipate that their use in biomedical MS proteomics research will offer actionable discoveries for the improvement of human health.