EtOH-LN cryoembedding workflow to minimize freezing artifact in frozen tissues: A pilot study in preparing tissues compatible with mass spectrometry-based spatial proteomics application.

Coolant-assisted liquid nitrogen (LN) flash freezing of frozen tissues has been widely adopted to preserve tissue morphology for histopathological annotations in mass spectrometry-based spatial proteomics techniques. However, existing coolants pose health risks upon inhalation and are expensive. To overcome this challenge, we present our pilot study by introducing the EtOH-LN workflow, which demonstrates the feasibility of using 95 % ethanol as a safer and easily accessible alternative to existing coolants for LN-based cryoembedding of frozen tissues. Our study reveals that both the EtOH-LN and LN-only cryoembedding workflows exhibit significantly reduced freezing artifacts compared to cryoembedding in cryostat (p < 0.005), while EtOH-LN (SD = 0.56) generates more consistent results compared to LN-only (SD = 1.29). We have modified a previously reported morphology restoration method to incorporate the EtOH-LN workflow, which successfully restored the tissue architecture from freezing artifacts (p < 0.05). Additional studies are required to validate the impact of the EtOH-LN workflow on the molecular profiles of tissues.

Tandem Affinity Purification (TAP) of Interacting Prey Proteins with FLAG- and HA-Tagged Bait Proteins.

Proteins often interact with each other to form complexes and play functional roles in almost all cellular processes. The study of protein-protein interactions is therefore critical to understand protein function and biological pathways. Affinity Purification coupled with Mass Spectrometry (AP-MS) is an invaluable technique for identifying the interaction partners in protein complexes. In this approach, the protein of interest is fused to an affinity tag, followed by the expression and purification of the fusion protein. The affinity-purified sample is then analyzed by mass spectrometry to identify the interaction partners of the bait proteins. In this chapter, we detail the protocol for tandem affinity purification (TAP) based on the use of the FLAG (a fusion tag with peptide sequence DYKDDDDK) and hemagglutinin (HA) peptide epitopes. The immunoprecipitation using dual-affinity tags offers the advantage of increasing the specificity of the purification with lower nonspecific-background interactions.

Identification and Quantification of Affinity-Purified Proteins with MaxQuant, Followed by the Discrimination of Nonspecific Interactions with the CRAPome Interface.

Affinity purification coupled to mass spectrometry (AP-MS) is a powerful method to analyze protein-protein interactions (PPIs). The AP-MS approach provides an unbiased analysis of the entire protein complex and is useful to identify indirect interactors. However, reliable protein identification from the complex AP-MS experiments requires appropriate control of false identifications and rigorous statistical analysis. Another challenge that can arise from AP-MS analysis is to distinguish bona fide interacting proteins from the non-specifically bound endogenous proteins or the "background contaminants" that co-purified by the bait experiments. In this chapter, we will first describe the protocol for performing in-solution trypsinization for the samples from the AP experiment followed by LC-MS/MS analysis. We will then detail the MaxQuant workflow for protein identification and quantification for the PPI data derived from the AP-MS experiment. Finally, we describe the CRAPome interface to process the data by filtering against contaminant lists, score the interactions and visualize the protein interaction networks.

A recent update on small-molecule kinase inhibitors for targeted cancer therapy and their therapeutic insights from mass spectrometry-based proteomic analysis.

Kinases are key regulatory signalling proteins governing numerous essential biological processes and cellular functions. Dysregulation of many protein kinases is associated with cancer initiation and progression. Given their crucial roles, there has been increasing interest in harnessing kinases as prospective drug targets for cancer. In recent decades, numerous small-molecule kinase inhibitors have been developed and revolutionized the cancer treatment landscape. Despite their great potential, challenges remain in developing highly selective and effective kinase inhibitors, with toxicity and resistance issues frequently arising. In this review, we first provide an overview of the role of kinases in carcinogenesis and describe the current progress with small-molecule kinase inhibitors that have been approved for clinical use. We then discuss the application of mass spectrometry (MS)-based proteomics strategies to help in the design of kinase inhibitors. Finally, we discuss the challenges and outlook concerning MS-based proteomics techniques for kinase drug research.

Comparison Between Dichloroacetate and Phenylbutyrate Treatment for Pyruvate Dehydrogenase Deficiency.

Pyruvate dehydrogenase (PDH) deficiency is caused by a number of pathogenic variants and the most common are found in the PDHA1 gene. The PDHA1 gene encodes one of the subunits of the PDH enzyme found in a carbohydrate metabolism pathway involved in energy production. Pathogenic variants of PDHA1 gene usually impact the α-subunit of PDH causing energy reduction. It potentially leads to increased mortality in sufferers. Potential treatments for this disease include dichloroacetate and phenylbutyrate, previously used for other diseases such as cancer and maple syrup urine disease. However, not much is known about their efficacy in treating PDH deficiency. Effective treatment for PDH deficiency is crucial as carbohydrate is needed in a healthy diet and rice is the staple food for a large portion of the Asian population. This review analysed the efficacy of dichloroacetate and phenylbutyrate as potential treatments for PDH deficiency caused by PDHA1 pathogenic variants. Based on the findings of this review, dichloroacetate will have an effect on most PDHA1 pathogenic variant and can act as a temporary treatment to reduce the lactic acidosis, a common symptom of PDH deficiency. Phenylbutyrate can only be used on patients with certain pathogenic variants (p.P221L, p.R234G, p.G249R, p.R349C, p.R349H) on the PDH protein. It is hoped that the review would provide an insight into these treatments and improve the quality of lives for patients with PDH deficiency.

Short open reading frames (sORFs) and microproteins: an update on their identification and validation measures.

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.

Regulation of signal transduction pathways in colorectal cancer: implications for therapeutic resistance.

Resistance to anti-cancer treatments is a critical and widespread health issue that has brought serious impacts on lives, the economy and public policies. Mounting research has suggested that a selected spectrum of patients with advanced colorectal cancer (CRC) tend to respond poorly to both chemotherapeutic and targeted therapeutic regimens. Drug resistance in tumours can occur in an intrinsic or acquired manner, rendering cancer cells insensitive to the treatment of anti-cancer therapies. Multiple factors have been associated with drug resistance. The most well-established factors are the emergence of cancer stem cell-like properties and overexpression of ABC transporters that mediate drug efflux. Besides, there is emerging evidence that signalling pathways that modulate cell survival and drug metabolism play major roles in the maintenance of multidrug resistance in CRC. This article reviews drug resistance in CRC as a result of alterations in the MAPK, PI3K/PKB, Wnt/ß-catenin and Notch pathways.

Molecular tissue profiling by MALDI imaging: recent progress and applications in cancer research.

Matrix-assisted laser desorption/ionization (MALDI) imaging is an emergent technology that has been increasingly adopted in cancer research. MALDI imaging is capable of providing global molecular mapping of the abundance and spatial information of biomolecules directly in the tissues without labeling. It enables the characterization of a wide spectrum of analytes, including proteins, peptides, glycans, lipids, drugs, and metabolites and is well suited for both discovery and targeted analysis. An advantage of MALDI imaging is that it maintains tissue integrity, which allows correlation with histological features. It has proven to be a valuable tool for probing tumor heterogeneity and has been increasingly applied to interrogate molecular events associated with cancer. It provides unique insights into both the molecular content and spatial details that are not accessible by other techniques, and it has allowed considerable progress in the field of cancer research. In this review, we first provide an overview of the MALDI imaging workflow and approach. We then highlight some useful applications in various niches of cancer research, followed by a discussion of the challenges, recent developments and future prospect of this technique in the field.

WIDENING THE BOTTLENECK OF PHOSPHOPROTEOMICS: EVOLVING STRATEGIES FOR PHOSPHOPEPTIDE ENRICHMENT.

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.

The evolution of two-dimensional gel electrophoresis - from proteomics to emerging alternative applications.

Two-dimensional gel electrophoresis (2-DE) is a technique that has been widely applied in a variety of proteomics studies. It is capable of resolving complex protein mixtures into individual protein spots based on their isoelectric point and molecular weight, enabling large-scale analysis of protein expression patterns for deciphering their changes in different biological conditions. 2-DE is a powerful tool that empowers researchers to perform differential qualitative and quantitative proteome analysis and is particularly advantageous for characterizing protein isoforms and post-translationally modified proteins. Despite its popularity as the workhorse for proteomics in the past few decades, it has been gradually displaced by the more sophisticated and high-performance mass spectrometry-based methods. However, there are several variations of the 2-DE technique that have emerged as promising approaches that shine new light on specific niches that 2-DE could still contribute. In this review, we first provide an overview of the applications of 2-DE, its merits and pitfalls in the current proteomic research arena, followed by a discussion on several alternative approaches for potential future applications.

Plasma/serum proteomics: depletion strategies for reducing high-abundance proteins for biomarker discovery.

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.

Proteogenomics: From next-generation sequencing (NGS) and mass spectrometry-based proteomics to precision medicine.

One of the best-established area within multi-omics is proteogenomics, whereby the underpinning technologies are next-generation sequencing (NGS) and mass spectrometry (MS). Proteogenomics has contributed significantly to genome (re)-annotation, whereby novel coding sequences (CDS) are identified and confirmed. By incorporating in-silico translated genome variants in protein database, single amino acid variants (SAAV) and splice proteoforms can be identified and quantified at peptide level. The application of proteogenomics in cancer research potentially enables the identification of patient-specific proteoforms, as well as the association of the efficacy or resistance of cancer therapy to different mutations. Here, we discuss how NGS/TGS data are analyzed and incorporated into the proteogenomic framework. These sequence data mainly originate from whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. We explain two major strategies for sequence analysis i.e., de novo assembly and reads mapping, followed by construction of customized protein databases using such data. Besides, we also elaborate on the procedures of spectrum to peptide sequence matching in proteogenomics, and the relationship between database size on the false discovery rate (FDR). Finally, we discuss the latest development in proteogenomics-assisted precision oncology and also challenges and opportunities in proteogenomics research.

Sudden Cardiac Death (SCD) - risk stratification and prediction with molecular biomarkers.

Sudden cardiac death (SCD) is a sudden, unexpected death that is caused by the loss of heart function. While SCD affects many patients suffering from coronary artery diseases (CAD) and heart failure (HF), a considerable number of SCD events occur in asymptomatic individuals. Certain risk factors for SCD have been identified and incorporated in different clinical scores, however, risk stratification using such algorithms is only useful for health management rather than for early detection and prediction of future SCD events in high-risk individuals. In this review, we discuss different molecular biomarkers that are used for early detection of SCD. This includes genetic biomarkers, where the majority of them are genomic variants for genes that encode for ion channels. Meanwhile, protein biomarkers often denote proteins that play roles in pathophysiological processes that lead to CAD and HF, notably (i) atherosclerosis that involves oxidative stress and inflammation, as well as (ii) cardiac tissue damage that involves neurohormonal and hemodynamic regulation and myocardial stress. Finally, we outline existing challenges and future directions including the use of OMICS strategy for biomarker discovery and the multimarker panels.

Probing the endogenous peptidomes of cancer for biomarkers: A new endeavor.

The life span of cancer patients can be prolonged with appropriate therapies if detected early. Mass screening for early detection of cancer, however, requires sensitive and specific biomarkers obtainable from body fluids such as blood or urine. To date, most biomarker discovery programs focus on the proteome rather than the endogenous peptidome. It has been long-established that tumor cells and stromal cells produce tumor resident proteases (TRPs) to remodel the surrounding tumor microenvironment in support of tumor progression. In fact, proteolytic products of TRPs have been shown to correlate with malignant behavior. Being of low molecular weight, these unique peptides can pass through the endothelial barrier of the vasculature into the bloodstream. As such, the cancer peptidome has increasingly become a focus for biomarker discovery. In this review, we discuss on the various aspects of the peptidome in cancer biomarker research.

Probing the colorectal cancer proteome for biomarkers: Current status and perspectives.

Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide. Biomarkers that can facilitate better clinical management of CRC are in high demand to improve patient outcome and to reduce mortality. In this regard, proteomic analysis holds a promising prospect in the hunt of novel biomarkers for CRC and in understanding the mechanisms underlying tumorigenesis. This review aims to provide an overview of the current progress of proteomic research, focusing on discovery and validation of diagnostic biomarkers for CRC. We will summarize the contributions of proteomic strategies to recent discoveries of protein biomarkers for CRC and also briefly discuss the potential and challenges of different proteomic approaches in biomarker discovery and translational applications.

Metaproteomic analysis of human gut microbiota: where are we heading?

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.

Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris.

Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS-PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection.