Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.

Proteomics, the large-scale study of all proteins of an organism or system, is a powerful tool for studying biological systems. It can provide a holistic view of the physiological and biochemical states of given samples through identification and quantification of large numbers of peptides and proteins. In forensic science, proteomics can be used as a confirmatory and orthogonal technique for well-built genomic analyses. Proteomics is highly valuable in cases where nucleic acids are absent or degraded, such as hair and bone samples. It can be used to identify body fluids, ethnic group, gender, individual, and estimate post-mortem interval using bone, muscle, and decomposition fluid samples. Compared to genomic analysis, proteomics can provide a better global picture of a sample. It has been used in forensic science for a wide range of sample types and applications. In this review, we briefly introduce proteomic methods, including sample preparation techniques, data acquisition using liquid chromatography-tandem mass spectrometry, and data analysis using database search, spectral library search, and de novo sequencing. We also summarize recent applications in the past decade of proteomics in forensic science with a special focus on human samples, including hair, bone, body fluids, fingernail, muscle, brain, and fingermark, and address the challenges, considerations, and future developments of forensic proteomics.

Glycation is a spontaneous post-translational modification of lysine, arginine, and the N-terminus of proteins. Protein glycation is closely related to the pathogenesis of human diseases, including diabetes, Alzheimer’s disease, renal disease, and cancer. The levels of advanced glycation end products (AGEs) are positively correlated with the progression of many diseases. However, it remains challenging to analyze glycation-related products, such as reactive carbonyl species, Schiff bases, Amadori compounds, and AGEs, because of their high heterogeneity. Many analysis methods, such as fluorescence detection, immunoassays, and liquid chromatography-tandem mass spectrometry, have attempted to correlate glycation products with diseases. Some enrichment methods have been used to increase the probability of detection of glycated proteins due to their low abundance in blood plasma. This review summarizes the enrichment and analysis methods that are currently used to identify glycation as a disease biomarker in exploratory studies.

Genetic information of reproduction and growth is essential for sustainable molluscan fisheries and aquaculture management. However, there is limited knowledge regarding the reproductive activity of the commercially important Pacific abalone Haliotis discus hannai. We performed de novo transcriptome sequencing of the ganglia in sexually immature and mature female Pacific abalone to better understand the sexual maturation process and the underlying molecular mechanisms. Of the ~305 million high-quality clean reads, 76,684 transcripts were de novo-assembled with an average length of 741 bp, 28.54% of which were annotated and classified according to Gene Ontology terms. There were 256 differentially expressed genes between the immature and mature abalone. Tandem mass spectrometry analysis, as compared to the predicted-peptide database of abalone ganglia transcriptome unigenes, identified 42 neuropeptide precursors, including 29 validated by peptidomic analyses. Label-free quantification revealed differential occurrences of 18 neuropeptide families between immature and mature abalone, including achatin, FMRFamide, crustacean cardioactive peptide, and pedal peptide A and B that were significantly more frequent at the mature stage. These results represent the first significant contribution to both maturation-related transcriptomic and peptidomic resources of the Pacific abalone ganglia and provide insight into the roles of various neuropeptides in reproductive regulation in marine gastropods.

The haptophyte alga Emiliania huxleyi is the most abundant coccolithophore in the modern ocean and produces elaborate calcite crystals, called coccolith, in a separate intracellular compartment known as the coccolith vesicle. Despite the importance of biomineralization in coccolithophores, the molecular mechanism underlying it remains unclear. Understanding this precise machinery at the molecular level will provide the knowledge needed to enable further manipulation of biomineralization. In our previous study, altering the calcium concentration modified the calcifying ability of E. huxleyi CCMP371. Therefore in this study, we tested E. huxleyi cells acclimated to three different calcium concentrations (0, 0.1, and 10 mM). To understand the whole transcript profile at different calcium concentrations, RNA-sequencing was performed and used for de novo assembly and annotation. The differentially expressed genes (DEGs) among the three different calcium concentrations were analyzed. The functional classification by gene ontology (GO) revealed that ‘intrinsic component of membrane’ was the most enriched of the GO terms at the ambient calcium concentration (10 mM) compared with the limited calcium concentrations (0 and 0.1 mM). Moreover, the DEGs in those comparisons were enriched mainly in ‘secondary metabolites biosynthesis, transport and catabolism’ and ‘signal transduction mechanisms’ in the KOG clusters and ‘processing in endoplasmic reticulum’, and ‘ABC transporters’ in the KEGG pathways. Furthermore, metabolic pathways involved in protein synthesis were enriched among the differentially expressed proteins. The results of this study provide a molecular profile for understanding the expression of transcripts and proteins in E. huxleyi at different calcium concentrations, which will help to identify the detailed mechanism of its calcification.