Quantitative Mass Spectrometry-Based Approaches in Cardiovascular Research

Abstract

ProteomicsC ardiovascular proteomics is a rapidly growing field, especially with the emergence of high-throughput mass spectrometry-based techniques. After the identification of proteins in a given sample, researchers are keen to find out how much of the protein is present, especially in a disease or a given state. Though challenging, over the past few years, several approaches have been established to quantify proteins from cells or tissues at a given state. In this review, we focus on mass spectrometry -based quantitative approaches for both large-scale and targeted proteomic analyses, which can be used in cardiovascular research. Several strategies using labeling and label-free approaches for both relative and absolute quantification (AQUA) have been established to meet the requirements of the researcher. Each of these methods has its own merits and limitations. We will discuss the methods that researchers can opt for to address the biological questions related to cardiovascular research.Cardiovascular proteomics is the branch of proteomics specifically applied to heart and vasculature. The field of proteomics has recently gained greater importance based on its potential to unravel the complex physiological and biochemical mechanisms and pathways in organisms and cells at the molecular level. The goal is to identify proteins that generate/mediate disease process, thereby, opening avenues to new diagnostics and therapeutic strategies. Proteins are much closer to the actual disease process, in most cases, than parent genes. They are the ultimate regulators of cell functions, and the vast majorities serve as drug targets. Differential protein expression analysis, by measuring the upregulated or downregulated proteins in disease state as compared with the normal healthy condition, is vital to understand the disease mechanism.In conjunction with various separation techniques, mass spectrometry (MS)-based methods evolved as the best techniques for the comprehensive characterization of proteins. 1,2After the initial protein identification, a more challenging approach in proteomics is the quantification of the proteins identified.3 Various methods of quantification by MS have been developed recently, for both relative and absolute measurements (Figure 1), and the technology is still expanding to find better and more efficient quantification approaches. This, in turn, is facilitated by the advancements in the MS instrumentation, including powerful analyzers and fragmentation technology. Most of the quantification methods are based on measurements at peptide level and fall under the bottom-up proteomics approach. In this review article, we will outline various MS-based approaches available for measuring the variation in protein abundances and how these techniques are playing a key role in cardiovascular research. Gel-Based QuantificationSeparation of proteins by polyacrylamide gel electrophoresis, followed by MS analysis, has been a common practice in proteomics. With the advent of 2D gel electrophoresis, quantificatio...

Comprehensive characterization of glioblastoma tumor tissues for biomarker identification using mass spectrometry-based label-free quantitative proteomics

“…The most common approach is by two-dimensional (2D) gel electrophoresis and 2D-differential in-gel electrophoresis (DIGE) for protein quantification followed by MS for protein identification. However, 2D gel-based approaches are limited by poor reproducibility, insufficient resolution of proteins, and low throughput (42). In recent years, several MS-based quantitative techniques have been developed for the direct comparison of protein expression levels between two or more different samples.…”

“…In recent years, several MS-based quantitative techniques have been developed for the direct comparison of protein expression levels between two or more different samples. Some of these require the incorporation of an isotopic label, either metabolically (SILAC), by chemical labeling (ICAT, ICPL), by enzymatic labeling (18O labeling), or with an isobaric tag (iTRAQ, TMT) (42). These methods have the following limitations: 1) metabolic labeling approaches require growth of cells in culture that preclude use with patient material; 2) problems arise in achieving high levels of labeling; and 3) greatly increased financial expense is associated with proprietary reagents.…”

The Path to Personalized Cardiovascular Medicine

“…[266][267][268] The advanced MS-based proteomic technologies enable the precise quantification of thousands of proteins and identification and mapping of their PTMs. 255,[269][270][271][272][273][274][275][276][277] Most proteins exert their functions through a variety of dynamic and highly regulated interactions with other proteins forming multiprotein complexes and signal transduction cascades. Coupling of various affinity purification methods to MS-based analysis (AP-MS) provides a powerful tool for the characterization of the protein interactome greatly advancing functional proteomics (see also Chapter 2).…”

Proteomic Analysis Reveals the Effect of Trichostatin A and Bone Marrow-Derived Dendritic Cells on the Fatty Acid Metabolism of NIH3T3 Cells under Oxygen–Glucose Deprivation Conditions

“…A global analysis of protein alterations may also help in understanding the potential mechanism of action of a drug . Recently, proteomics has been used to study CVDs. − In this study, we used a model of OGD, − which is widely accepted as mimicking the conditions present during MI, and performed a proteomic analysis to analyze the protein expression changes in NIH3T3 mouse fibroblasts to explore the mechanism of action of TSA and bone marrow-derived dendritic cells (BMDCs) in these cells under OGD conditions.…”