comprehensive-workflow-mass-spectrometry-based-shotgun-proteomics

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

quantitative-proteomics-workflow-using-multiple-reaction-monitoring

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Indian Institute of Technology Bombay

After a successful completion of the Human Genome Project, deciphering the mystery surrounding the human proteome posed a major challenge. Despite not being largely involved in the Human Genome Project, the Indian scientific community contributed towards proteomic research along with the global community. Currently, more than 76 research/academic institutes and nearly 145 research labs are involved in core proteomic research across India. The Indian researchers have been major contributors in drafting the "human proteome map" along with international efforts. In addition to this, virtual proteomics labs, proteomics courses and remote triggered proteomics labs have helped to overcome the limitations of proteomics education posed due to expensive lab infrastructure. The establishment of Proteomics Society, India (PSI) has created a platform for the Indian proteomic researchers to share ideas, research collaborations and conduct annual conferences and workshops. Indian proteomic research is really moving forward with the global proteomics community in a quest to solve the mysteries of proteomics. A draft map of the human proteome enhances the enthusiasm among intellectuals to promote proteomic research in India to the world.This article is part of a Special Issue entitled: Proteomics in India.

Proteomics research in India: an update.

Proteomics is at the epicenter of post-genomics biotechnologies that are currently driving the next generation system science. Moreover, proteomics is a truly global science. The 6(th) Annual Meeting of Proteomics Society, India (PSI) and International Conference on "Proteomics from Discovery to Function" held from December 7-9, 2014, was a transformative endeavor for global proteomics, bringing together the luminaries in the field of proteomics for the very first time in India. This meeting report presents the lessons learned and the highlights of this international scientific conference that was comprised of nine thematic sessions, pre- and post-conference workshops, and an opportunity to cultivate enduring collaborations for proteomics science to benefit both India and global society. The conference had an unforgettable impression on the participants: for the first time, India hosted past and present President and Council members from the Human Proteome Organization (HUPO), along with eminent scientists and young scholars from India and abroad in the field of proteomics at such a large scale, a major highlight of this international event. In all, the PSI 2014 was a milestone conference that has firmly poised the Indian life sciences community as a leading contributor to post-genomics life sciences, thus cultivating crucial trans-generational capacity and inspiration by recognizing the emerging scholars and omics systems scientists who can think and conduct science from cell to society. 

Meeting Report: "Proteomics from Discovery to Function:" 6th Annual Meeting of Proteomics Society, India and International Conference-A Milestone for the Indian Proteomics Community.

Targeted Proteomics Workshop and International Symposium (IIT Bombay, Mumbai, India): An Accelerator for Global Proteomics.

Gliomas are heterogeneous and most commonly occurring brain tumors. Blood-brain barrier restricts the entry of brain tumor proteins into blood stream thus limiting the usage of serum or plasma for proteomic analysis. Our study aimed at understanding the molecular basis of aggressiveness of various grades of brain tumors using isobaric tagging for relative and absolute quantification (iTRAQ) based mass spectrometry. Tissue proteomic analysis of various grades of gliomas was performed using four-plex iTRAQ. We labeled five sets (each set consists of control, grade-II, III, and IV tumor samples) of individual glioma patients using iTRAQ reagents. Significantly altered proteins were subjected to bioinformatics analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID). Various metabolic pathways like glycolysis, TCA-cycle, electron transport chain, lactate metabolism, and blood coagulation pathways were majorly observed to be perturbed in gliomas. Most of the identified proteins involved in redox reactions, protein folding, pre-messenger RNA (mRNA) processing, antiapoptosis, and blood coagulation were found to be upregulated in gliomas. Transcriptomics data of glioblastoma multiforme (GBM), low-grade gliomas (LGGs), and controls were downloaded from The Cancer Genome Atlas (TCGA) data portal and further analyzed using BRB-Array tools. Expression levels of a few significantly altered proteins like lactate dehydrogenase, alpha-1 antitrypsin, fibrinogen alpha chain, nucleophosmin, annexin A5, thioredoxin, ferritin light chain, thymosin beta-4-like protein 3, superoxide dismutase-2, and peroxiredoxin-1 and 6 showed a positive correlation with increasing grade of gliomas thereby offering an insight into molecular basis behind their aggressive nature. Several proteins identified in different grades of gliomas are potential grade-specific markers, and perturbed pathways provide comprehensive overview of molecular cues involved in glioma pathogenesis.

Tissue Proteome Analysis of Different Grades of Human Gliomas Provides Major Cues for Glioma Pathogenesis.

Heterogeneity within GBMs and variability of visualized fluorescence combine to confer practical limitations to the technique of optical imaging. A biometric analysis was planned to objectively ascertain and analyse this phenomenon METHODS: 25 adult glioblastoma subjects undergoing resection were prospectively accrued. Biopsies were taken from various parts of the tumor and safe peritumoral zones. White light (WL) and visualized fluorescence was subjectively recorded. Corresponding histopathology [coalescent (C) or infiltrating (I) tumor] and protoporphyrin-IX (PPIX) levels were assayed.

Objective assessment of intraoperative tumor fluorescence reveals biological heterogeneity within glioblastomas: a biometric study.

: Despite being rare cancers, gliomas account for a significant number of cancer-related deaths. Identification and treatment of these tumors at an early stage would greatly improve the therapeutic outcomes. There is an urgent need for diagnostic and prognostic markers, which can identify disease early and discriminate the subtypes of these tumors thereby improving the existing treatment modalities.: In this article, we have reviewed published literature on proteomics biomarkers for gliomas and their importance in diagnosis or prognosis. Proteomic studies for the discovery of protein, autoantibody biomarkers, and biological pathway alterations in serum, CSF and tumor biopsies have been discussed in this review.: The rapid development in the field of mass spectrometry and increased sensitivity and reproducibility in assays has led to the identification and quantification of large number of proteins very precisely. Though genomic markers are the prime focus in the classification of gliomas, incorporating protein markers would further improve the existing classification. In this regard, data mining and studies on large cohorts of glioma patients would help in the identification of diagnostic and prognostic markers ultimately translating to the clinics.

Glioma tumor proteomics: clinically useful protein biomarkers and future perspectives.

Blood plasma is one of the most widely used samples for cancer biomarker discovery research as well as clinical investigations for diagnostic and therapeutic purposes. However, the plasma proteome is extremely complex due to its wide dynamic range of protein concentrations and the presence of high-abundance proteins. Here we have described an optimized, integrated quantitative proteomics pipeline combining the label-free and multiplexed-labeling-based (iTRAQ and TMT) plasma proteome profiling methods for biomarker discovery, followed by the targeted approaches for validation of the identified potential marker proteins. In this workflow, the targeted quantitation of proteins is carried out by multiple-reaction monitoring (MRM) and parallel-reaction monitoring (PRM) mass spectrometry. Thus, our approach enables both unbiased screenings of biomarkers and their subsequent selective validation in human plasma. The overall procedure takes only ~2 days to complete, including the time for data acquisition (excluding database searching). This protocol is quick, flexible, and eliminates the need for a separate immunoassay-based validation workflow in blood cancer biomarker investigations. We anticipate that this plasma proteomics workflow will help to accelerate the cancer biomarker discovery program and provide a valuable resource to the cancer research community.

An Integrated Quantitative Proteomics Workflow for Cancer Biomarker Discovery and Validation in Plasma.

The altered molecular proteins and pathways in response to COVID-19 infection are still unclear. Here, we performed a comprehensive proteomics-based investigation of nasopharyngeal swab samples from patients with COVID-19 to study the host response by employing simple extraction strategies. Few of the host proteins such as interleukin-6, L-lactate dehydrogenase, C-reactive protein, Ferritin, and aspartate aminotransferase were found to be upregulated only in COVID-19-positive patients using targeted multiple reaction monitoring studies. The most important pathways identified by enrichment analysis were neutrophil degranulation, interleukin-12 signaling pathways, and mRNA translation of proteins thus providing the detailed investigation of host response in COVID-19 infection. Thus, we conclude that mass spectrometry-detected host proteins have a potential for disease severity progression; however, suitable validation strategies should be deployed for the clinical translation. Furthermore, the  docking of potential drugs with host proteins involved in the interleukin-12 signaling pathway might aid in COVID-19 therapeutic interventions.

Proteomic investigation reveals dominant alterations of neutrophil degranulation and mRNA translation pathways in patients with COVID-19.

The pestilential pathogen SARS-CoV-2 has led to a seemingly ceaseless pandemic of COVID-19. The healthcare sector is under a tremendous burden, thus necessitating the prognosis of COVID-19 severity. This in-depth study of plasma proteome alteration provides insights into the host physiological response towards the infection and also reveals the potential prognostic markers of the disease. Using label-free quantitative proteomics, we performed deep plasma proteome analysis in a cohort of 71 patients (20 COVID-19 negative, 18 COVID-19 non-severe, and 33 severe) to understand the disease dynamics. Of the 1200 proteins detected in the patient plasma, 38 proteins were identified to be differentially expressed between non-severe and severe groups. The altered plasma proteome revealed significant dysregulation in the pathways related to peptidase activity, regulated exocytosis, blood coagulation, complement activation, leukocyte activation involved in immune response, and response to glucocorticoid biological processes in severe cases of SARS-CoV-2 infection. Furthermore, we employed supervised machine learning (ML) approaches using a linear support vector machine model to identify the classifiers of patients with non-severe and severe COVID-19. The model used a selected panel of 20 proteins and classified the samples based on the severity with a classification accuracy of 0.84. Putative biomarkers such as angiotensinogen and SERPING1 and ML-derived classifiers including the apolipoprotein B, SERPINA3, and fibrinogen gamma chain were validated by targeted mass spectrometry-based multiple reaction monitoring (MRM) assays. We also employed an  screening approach against the identified target proteins for the therapeutic management of COVID-19. We shortlisted two FDA-approved drugs, namely, selinexor and ponatinib, which showed the potential of being repurposed for COVID-19 therapeutics. Overall, this is the first most comprehensive plasma proteome investigation of COVID-19 patients from the Indian population, and provides a set of potential biomarkers for the disease severity progression and targets for therapeutic interventions.

Proteomics and Machine Learning Approaches Reveal a Set of Prognostic Markers for COVID-19 Severity With Drug Repurposing Potential.

The emergence of omics technologies over the last decade has helped in advancement of research and our understanding of complex diseases like brain cancers. However, barring genomics, no other omics technology has been able to find utility in clinical settings. The recent advancements in mass spectrometry instrumentation have resulted in proteomics technologies becoming more sensitive and reliable. Targeted proteomics, a relatively new branch of mass spectrometry-based proteomics has shown immense potential in addressing the shortcomings of the standard molecular biology-based techniques like Western blotting and Immunohistochemistry. In this study we demonstrate the utility of Multiple reaction monitoring (MRM), a targeted proteomics approach, in quantifying peptides from proteins like Apolipoprotein A1 (APOA1), Apolipoprotein E (APOE), Prostaglandin H2 D-Isomerase (PTGDS), Vitronectin (VTN) and Complement C3 (C3) in cerebrospinal fluid (CSF) collected from Glioma and Meningioma patients. Additionally, we also report transitions for peptides from proteins - Vimentin (VIM), Cystatin-C (CST3) and Clusterin (CLU) in surgically resected Meningioma tissues; Annexin A1 (ANXA1), Superoxide dismutase (SOD2) and VIM in surgically resected Glioma tissues; and Microtubule associated protein-2 (MAP-2), Splicing factor 3B subunit 2 (SF3B2) and VIM in surgically resected Medulloblastoma tissues. To our knowledge, this is the first study reporting the use of MRM to validate proteins from three types of brain malignancies and two different bio-specimens. Future studies involving a large cohort of samples aimed at accurately detecting and quantifying peptides of proteins with roles in brain malignancies could potentially result in a panel of proteins showing ability to classify and grade tumors. Successful application of these techniques could ultimately offer alternative strategies with increased accuracy, sensitivity and lower turnaround time making them translatable to the clinics.

ArticleTotal : 2	Year
Publication title Cited by 8	2021
Publication title Cited by 3	2021

Article	Year
Proteomics research in India: an update. Journal of proteomics\| PubMed ID: 25868663	2015
Meeting Report: "Proteomics from Discovery to Function:" 6th Annual Meeting of Proteomics Society, India and International Conference-A Milestone for the Indian Proteomics Community. Omics : a journal of integrative biology\| PubMed ID: 26415138	2015
Targeted Proteomics Workshop and International Symposium (IIT Bombay, Mumbai, India): An Accelerator for Global Proteomics. Omics : a journal of integrative biology\| PubMed ID: 27093106	2016
Tissue Proteome Analysis of Different Grades of Human Gliomas Provides Major Cues for Glioma Pathogenesis. Omics : a journal of integrative biology\| PubMed ID: 28481733	2017
Objective assessment of intraoperative tumor fluorescence reveals biological heterogeneity within glioblastomas: a biometric study. Journal of neuro-oncology\| PubMed ID: 32020478	2020
Glioma tumor proteomics: clinically useful protein biomarkers and future perspectives. Expert review of proteomics\| PubMed ID: 32067544	2020
An Integrated Quantitative Proteomics Workflow for Cancer Biomarker Discovery and Validation in Plasma. Frontiers in oncology\| PubMed ID: 33072574	2020
Proteomic investigation reveals dominant alterations of neutrophil degranulation and mRNA translation pathways in patients with COVID-19. iScience\| PubMed ID: 33558857	2021
Proteomics and Machine Learning Approaches Reveal a Set of Prognostic Markers for COVID-19 Severity With Drug Repurposing Potential. Frontiers in physiology\| PubMed ID: 33995121	2021
Multiple Reaction Monitoring-Based Targeted Assays for the Validation of Protein Biomarkers in Brain Tumors. Frontiers in oncology\| PubMed ID: 34055594	2021

Saicharan Ghantasala

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