Phosphorylation is a form of protein posttranslational modification (PTM) that regulates many biological processes. Whereas phosphoproteomics is a scientific discipline that identifies and quantifies the phosphorylated proteome using mass spectrometry (MS). This task is extremely challenging as ~30% of the human proteome is phosphorylated; and each phosphoprotein may exist as multiple phospho‐isoforms that are present in low abundance and stoichiometry. Hence, phosphopeptide enrichment techniques are indispensable to (phospho)proteomics laboratories. These enrichment methods encompass widely‐adopted techniques such as (i) affinity‐based chromatography; (ii) ion exchange and mixed‐mode chromatography (iii) enrichment with phospho‐specific antibodies and protein domains, and (iv) functionalized polymers and other less common but emerging technologies such as hydroxyapatite chromatography and precipitation with inorganic ions. Here, we review these techniques, their history, continuous development and evaluation. Besides, we outline associating challenges of phosphoproteomics that are linked to experimental design, sample preparation, and proteolytic digestion. In addition, we also discuss about the future outlooks in phosphoproteomics, focusing on elucidating the noncanonical phosphoproteome and deciphering the “dark phosphoproteome”. © 2020 John Wiley & Sons Ltd.

Plasma and serum are widely used for proteomics-based biomarker discovery. However, analysis of these biofluids is highly challenging due to the complexity and wide dynamic range of their proteomes. Notably, highly abundant proteins tend to obscure the detection of potential biomarkers that are usually of lower concentrations. Among the strategies to resolve this problem are: depletion of high-abundance proteins, enrichment of low abundant proteins of interest and prefractionation. In this review, we focus on current and emerging depletion techniques used to enhance the detection and identification of the less abundant proteins in plasma and serum. We discuss the applications and contributions of these methods to proteomics analysis of plasma and serum alongside their limitations and future perspectives.

The human gut is home to complex microbial populations that change dynamically in response to various internal and external stimuli. The gut microbiota provides numerous functional benefits that are crucial for human health but in the setting of a disturbed equilibrium, the microbial community can cause deleterious outcomes such as diseases and cancers. Characterization of the functional activities of human gut microbiota is fundamental to understand their roles in human health and disease. Metaproteomics, which refers to the study of the entire protein collection of the microbial community in a given sample is an emerging area of research that provides informative details concerning functional aspects of the microbiota. In this mini review, we present a summary of the progress of metaproteomic analysis for studying the functional role of gut microbiota. This is followed by an overview of the experimental approaches focusing on fecal specimen for metaproteomics and is concluded by a discussion on the challenges and future directions of metaproteomic research.

A short open reading frame (sORFs) constitutes ≤ 300 bases, encoding a microprotein or sORF-encoded protein (SEP) which comprises ≤ 100 amino acids. Traditionally dismissed by genome annotation pipelines as meaningless noise, sORFs were found to possess coding potential with ribosome profiling (RIBO-Seq), which unveiled sORF-based transcripts at various genome locations. Nonetheless, the existence of corresponding microproteins that are stable and functional was little substantiated by experimental evidence initially. With recent advancements in multi-omics, the identification, validation, and functional characterisation of sORFs and microproteins have become feasible. In this review, we discuss the history and development of an emerging research field of sORFs and microproteins. In particular, we focus on an array of bioinformatics and OMICS approaches used for predicting, sequencing, validating, and characterizing these recently discovered entities. These strategies include RIBO-Seq which detects sORF transcripts via ribosome footprints, and mass spectrometry (MS)-based proteomics for sequencing the resultant microproteins. Subsequently, our discussion extends to the functional characterisation of microproteins by incorporating CRISPR/Cas9 screen and protein–protein interaction (PPI) studies. Our review discusses not only detection methodologies, but we also highlight on the challenges and potential solutions in identifying and validating sORFs and their microproteins. The novelty of this review lies within its validation for the functional role of microproteins, which could contribute towards the future landscape of microproteomics.

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