Deciphering a proteomic signature for the early detection of breast cancer from breast milk: the role of quantitative proteomics.

INTRODUCTION: Breast cancer is one of the most prevalent cancers among women in the United States. Current research regarding breast milk has been focused on the composition and its role in infant growth and development. There is little information about the proteins, immune cells, and epithelial cells present in breast milk which can be indicative of the emergence of BC cells and tumors. AREAS COVERED: We summarize all breast milk studies previously done in our group using proteomics. These studies include 1D-PAGE and 2D-PAGE analysis of breast milk samples, which include within woman and across woman comparisons to identify dysregulated proteins in breast milk and the roles of these proteins in both the development of BC and its diagnosis. Our projected outlook for the use of milk for cancer detection is also discussed. EXPERT OPINION: Analyzing the samples by multiple methods allows one to interrogate a set of samples with various biochemical methods that complement each other, thus providing a more comprehensive proteome. Complementing methods like 1D-PAGE, 2D-PAGE, in-solution digestion and proteomics analysis with PTM-omics, peptidomics, degradomics, or interactomics will provide a better understanding of the dysregulated proteins, but also the modifications or interactions between these proteins.

A Nanorobotics-Based Approach of Breast Cancer in the Nanotechnology Era.

We are living in an era of advanced nanoscience and nanotechnology. Numerous nanomaterials, culminating in nanorobots, have demonstrated ingenious applications in biomedicine, including breast cancer (BC) nano-theranostics. To solve the complicated problem of BC heterogeneity, non-targeted drug distribution, invasive diagnostics or surgery, resistance to classic onco-therapies and real-time monitoring of tumors, nanorobots are designed to perform multiple tasks at a small scale, even at the organelles or molecular level. Over the last few years, most nanorobots have been bioengineered as biomimetic and biocompatible nano(bio)structures, resembling different organisms and cells, such as urchin, spider, octopus, fish, spermatozoon, flagellar bacterium or helicoidal cyanobacterium. In this review, readers will be able to deepen their knowledge of the structure, behavior and role of several types of nanorobots, among other nanomaterials, in BC theranostics. We summarized here the characteristics of many functionalized nanodevices designed to counteract the main neoplastic hallmark features of BC, from sustaining proliferation and evading anti-growth signaling and resisting programmed cell death to inducing angiogenesis, activating invasion and metastasis, preventing genomic instability, avoiding immune destruction and deregulating autophagy. Most of these nanorobots function as targeted and self-propelled smart nano-carriers or nano-drug delivery systems (nano-DDSs), enhancing the efficiency and safety of chemo-, radio- or photodynamic therapy, or the current imagistic techniques used in BC diagnosis. Most of these nanorobots have been tested in vitro, using various BC cell lines, as well as in vivo, mainly based on mice models. We are still waiting for nanorobots that are low-cost, as well as for a wider transition of these favorable effects from laboratory to clinical practice.

Biological Basis of Breast Cancer-Related Disparities in Precision Oncology Era.

Precision oncology is based on deep knowledge of the molecular profile of tumors, allowing for more accurate and personalized therapy for specific groups of patients who are different in disease susceptibility as well as treatment response. Thus, onco-breastomics is able to discover novel biomarkers that have been found to have racial and ethnic differences, among other types of disparities such as chronological or biological age-, sex/gender- or environmental-related ones. Usually, evidence suggests that breast cancer (BC) disparities are due to ethnicity, aging rate, socioeconomic position, environmental or chemical exposures, psycho-social stressors, comorbidities, Western lifestyle, poverty and rurality, or organizational and health care system factors or access. The aim of this review was to deepen the understanding of BC-related disparities, mainly from a biomedical perspective, which includes genomic-based differences, disparities in breast tumor biology and developmental biology, differences in breast tumors' immune and metabolic landscapes, ecological factors involved in these disparities as well as microbiomics- and metagenomics-based disparities in BC. We can conclude that onco-breastomics, in principle, based on genomics, proteomics, epigenomics, hormonomics, metabolomics and exposomics data, is able to characterize the multiple biological processes and molecular pathways involved in BC disparities, clarifying the differences in incidence, mortality and treatment response for different groups of BC patients.

Onco-Breastomics: An Eco-Evo-Devo Holistic Approach.

Known as a diverse collection of neoplastic diseases, breast cancer (BC) can be hyperbolically characterized as a dynamic pseudo-organ, a living organism able to build a complex, open, hierarchically organized, self-sustainable, and self-renewable tumor system, a population, a species, a local community, a biocenosis, or an evolving dynamical ecosystem (i.e., immune or metabolic ecosystem) that emphasizes both developmental continuity and spatio-temporal change. Moreover, a cancer cell community, also known as an oncobiota, has been described as non-sexually reproducing species, as well as a migratory or invasive species that expresses intelligent behavior, or an endangered or parasite species that fights to survive, to optimize its features inside the host's ecosystem, or that is able to exploit or to disrupt its host circadian cycle for improving the own proliferation and spreading. BC tumorigenesis has also been compared with the early embryo and placenta development that may suggest new strategies for research and therapy. Furthermore, BC has also been characterized as an environmental disease or as an ecological disorder. Many mechanisms of cancer progression have been explained by principles of ecology, developmental biology, and evolutionary paradigms. Many authors have discussed ecological, developmental, and evolutionary strategies for more successful anti-cancer therapies, or for understanding the ecological, developmental, and evolutionary bases of BC exploitable vulnerabilities. Herein, we used the integrated framework of three well known ecological theories: the Bronfenbrenner's theory of human development, the Vannote's River Continuum Concept (RCC), and the Ecological Evolutionary Developmental Biology (Eco-Evo-Devo) theory, to explain and understand several eco-evo-devo-based principles that govern BC progression. Multi-omics fields, taken together as onco-breastomics, offer better opportunities to integrate, analyze, and interpret large amounts of complex heterogeneous data, such as various and big-omics data obtained by multiple investigative modalities, for understanding the eco-evo-devo-based principles that drive BC progression and treatment. These integrative eco-evo-devo theories can help clinicians better diagnose and treat BC, for example, by using non-invasive biomarkers in liquid-biopsies that have emerged from integrated omics-based data that accurately reflect the biomolecular landscape of the primary tumor in order to avoid mutilating preventive surgery, like bilateral mastectomy. From the perspective of preventive, personalized, and participatory medicine, these hypotheses may help patients to think about this disease as a process governed by natural rules, to understand the possible causes of the disease, and to gain control on their own health.

Proteomics analysis of human breast milk by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry to assess breast cancer risk.

Breast cancer (BC) is one of the most common cancers and one of the most common causes for cancer-related mortality. Discovery of protein biomarkers associated with cancer is considered important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)-based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregulations of breast milk proteins in comparison pairs of BC versus control. These dysregulated proteins might be considered potential future biomarkers of BC. Identification of potential biomarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in different sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed 2D-PAGE coupled with nano-liquid chromatography-tandem MS (nanoLC-MS/MS) in a small-scale study on a set of six human breast milk pairs (three BC samples vs. three controls) and we identified several dysregulated proteins that have potential roles in cancer progression and might be considered potential BC biomarkers in the future.

Two-Dimensional Polyacrylamide Gel Electrophoresis Coupled with Nanoliquid Chromatography-Tandem Mass Spectrometry-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in the MCF7 Breast Cancer Cell Line Transfected for Jumping Translocation Breakpoint Protein Overexpression.

The identification of new genes/proteins involved in breast cancer (BC) occurrence is widely used to discover novel biomarkers and understand the molecular mechanisms of BC initiation and progression. The jumping translocation breakpoint (JTB) gene may act both as a tumor suppressor or oncogene in various types of tumors, including BC. Thus, the JTB protein could have the potential to be used as a biomarker in BC, but its neoplastic mechanisms still remain unknown or controversial. We previously analyzed the interacting partners of JTBhigh protein extracted from transfected MCF7 BC cell line using SDS-PAGE complemented with in-solution digestion, respectively. The previous results suggested the JTB contributed to the development of a more aggressive phenotype and behavior for the MCF7 BC cell line through synergistic upregulation of epithelial-mesenchymal transition (EMT), mitotic spindle, and fatty acid metabolism-related pathways. In this work, we aim to complement the previously reported JTB proteomics-based experiments by investigating differentially expressed proteins (DEPs) and tumorigenic pathways associated with JTB overexpression using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Statistically different gel spots were picked for protein digestion, followed by nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis. We identified six DEPs related to the JTBhigh condition vs. control that emphasize a pro-tumorigenic (PT) role. Twenty-one proteins, which are known to be usually overexpressed in cancer cells, emphasize an anti-tumorigenic (AT) role when low expression occurs. According to our previous results, proteins that have a PT role are mainly involved in the activation of the EMT process. Interestingly, JTB overexpression has been correlated here with a plethora of significant upregulated and downregulated proteins that sustain JTB tumor suppressive functions. Our present and previous results sustain the necessity of the complementary use of different proteomics-based methods (SDS-PAGE, 2D-PAGE, and in-solution digestion) followed by tandem mass spectrometry to avoid their limitations, with each method leading to the delineation of specific clusters of DEPs that may be merged for a better understanding of molecular pathways and neoplastic mechanisms related to the JTB's role in BC initiation and progression.

Proteomics Analysis of Lymphoblastoid Cell Lines from Patients with Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) consists of the progressive degeneration of motor neurons, caused by poorly understood mechanisms for which there is no cure. Some of the cellular perturbations associated with ALS can be detected in peripheral cells, including lymphocytes from blood. A related cell system that is very suitable for research consists of human lymphoblastoid cell lines (LCLs), which are immortalized lymphocytes. LCLs that can be easily expanded in culture and can be maintained for long periods as stable cultures. We investigated, on a small set of LCLs, if a proteomics analysis using liquid chromatography followed by tandem mass spectrometry reveals proteins that are differentially present in ALS versus healthy controls. We found that individual proteins, the cellular and molecular pathways in which these proteins participate, are detected as differentially present in the ALS samples. Some of these proteins and pathways are already known to be perturbed in ALS, while others are new and present interest for further investigations. These observations suggest that a more detailed proteomics analysis of LCLs, using a larger number of samples, represents a promising approach for investigating ALS mechanisms and to search for therapeutic agents. Proteomics data are available via ProteomeXchange with identifier PXD040240.

Omics-Based Investigations of Breast Cancer.

Breast cancer (BC) is characterized by an extensive genotypic and phenotypic heterogeneity. In-depth investigations into the molecular bases of BC phenotypes, carcinogenesis, progression, and metastasis are necessary for accurate diagnoses, prognoses, and therapy assessments in predictive, precision, and personalized oncology. This review discusses both classic as well as several novel omics fields that are involved or should be used in modern BC investigations, which may be integrated as a holistic term, onco-breastomics. Rapid and recent advances in molecular profiling strategies and analytical techniques based on high-throughput sequencing and mass spectrometry (MS) development have generated large-scale multi-omics datasets, mainly emerging from the three "big omics", based on the central dogma of molecular biology: genomics, transcriptomics, and proteomics. Metabolomics-based approaches also reflect the dynamic response of BC cells to genetic modifications. Interactomics promotes a holistic view in BC research by constructing and characterizing protein-protein interaction (PPI) networks that provide a novel hypothesis for the pathophysiological processes involved in BC progression and subtyping. The emergence of new omics- and epiomics-based multidimensional approaches provide opportunities to gain insights into BC heterogeneity and its underlying mechanisms. The three main epiomics fields (epigenomics, epitranscriptomics, and epiproteomics) are focused on the epigenetic DNA changes, RNAs modifications, and posttranslational modifications (PTMs) affecting protein functions for an in-depth understanding of cancer cell proliferation, migration, and invasion. Novel omics fields, such as epichaperomics or epimetabolomics, could investigate the modifications in the interactome induced by stressors and provide PPI changes, as well as in metabolites, as drivers of BC-causing phenotypes. Over the last years, several proteomics-derived omics, such as matrisomics, exosomics, secretomics, kinomics, phosphoproteomics, or immunomics, provided valuable data for a deep understanding of dysregulated pathways in BC cells and their tumor microenvironment (TME) or tumor immune microenvironment (TIMW). Most of these omics datasets are still assessed individually using distinct approches and do not generate the desired and expected global-integrative knowledge with applications in clinical diagnostics. However, several hyphenated omics approaches, such as proteo-genomics, proteo-transcriptomics, and phosphoproteomics-exosomics are useful for the identification of putative BC biomarkers and therapeutic targets. To develop non-invasive diagnostic tests and to discover new biomarkers for BC, classic and novel omics-based strategies allow for significant advances in blood/plasma-based omics. Salivaomics, urinomics, and milkomics appear as integrative omics that may develop a high potential for early and non-invasive diagnoses in BC. Thus, the analysis of the tumor circulome is considered a novel frontier in liquid biopsy. Omics-based investigations have applications in BC modeling, as well as accurate BC classification and subtype characterization. The future in omics-based investigations of BC may be also focused on multi-omics single-cell analyses.

Two-Dimensional-PAGE Coupled with nLC-MS/MS-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in MCF7 Breast Cancer Cells Transfected for JTB Protein Silencing.

The identification of new cancer-associated genes/proteins, the characterization of their expression variation, the interactomics-based assessment of differentially expressed genes/proteins (DEGs/DEPs), and understanding the tumorigenic pathways and biological processes involved in BC genesis and progression are necessary and possible by the rapid and recent advances in bioinformatics and molecular profiling strategies. Taking into account the opinion of other authors, as well as based on our own team's in vitro studies, we suggest that the human jumping translocation breakpoint (hJTB) protein might be considered as a tumor biomarker for BC and should be studied as a target for BC therapy. In this study, we identify DEPs, carcinogenic pathways, and biological processes associated with JTB silencing, using 2D-PAGE coupled with nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics applied to a MCF7 breast cancer cell line, for complementing and completing our previous results based on SDS-PAGE, as well as in-solution proteomics of MCF7 cells transfected for JTB downregulation. The functions of significant DEPs are analyzed using GSEA and KEGG analyses. Almost all DEPs exert pro-tumorigenic effects in the JTBlow condition, sustaining the tumor suppressive function of JTB. Thus, the identified DEPs are involved in several signaling and metabolic pathways that play pro-tumorigenic roles: EMT, ERK/MAPK, PI3K/AKT, Wnt/ß-catenin, mTOR, C-MYC, NF-κB, IFN-γ and IFN-α responses, UPR, and glycolysis/gluconeogenesis. These pathways sustain cancer cell growth, adhesion, survival, proliferation, invasion, metastasis, resistance to apoptosis, tight junctions and cytoskeleton reorganization, the maintenance of stemness, metabolic reprogramming, survival in a hostile environment, and sustain a poor clinical outcome. In conclusion, JTB silencing might increase the neoplastic phenotype and behavior of the MCF7 BC cell line. The data is available via ProteomeXchange with the identifier PXD046265.

Proteomic analysis of the lake trout (Salvelinus namaycush) heart and blood: The beginning of a comprehensive lake trout protein database.

Lake trout (Salvelinus namaycush) are a top-predator species in the Laurentian Great Lakes that are often used as bioindicators of chemical stressors in the ecosystem. Although many studies are done using these fish to determine concentrations of stressors like legacy persistent, bioaccumulative and toxic chemicals, there are currently no proteomic studies on the biological effects these stressors have on the ecosystem. This lack of proteomic studies on Great Lakes lake trout is because there is currently no complete, comprehensive protein database for this species. Here, we employed proteomics approaches to develop a lake trout protein database that could aid in future research on this fish, in particular exposomics and adductomics. The current study utilized heart tissue and blood from two lake trout. Our previous work using lake trout liver revealed 4194 potential protein hits in the NCBI databases and 3811 potential protein hits in the UniProtKB databases. In the current study, using the NCBI databases we identified 838 proteins for the heart and 580 proteins for the blood tissues in the biological replicate 1 (BR1) and 1180 potential protein hits for the heart and 561 potential protein hits for the blood in BR2. Similar results were obtained using the UniProtKB databases. This study builds on our previous work by continuing to build the first comprehensive lake trout protein database and provides insight into protein homology through evolutionary relationships. This data is available via the PRIDE partner repository with the dataset identifier PXD023970.

Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research.

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.

Investigating the Function of Human Jumping Translocation Breakpoint Protein (hJTB) and Its Interacting Partners through In-Solution Proteomics of MCF7 Cells.

Human jumping translocation breakpoint (hJTB) gene is located on chromosome 1q21 and is involved in unbalanced translocation in many types of cancer. JTB protein is ubiquitously present in normal cells but it is found to be overexpressed or downregulated in various types of cancer cells, where this protein and its isoforms promote mitochondrial dysfunction, resistance to apoptosis, genomic instability, proliferation, invasion and metastasis. Hence, JTB could be a tumor biomarker for different types of cancer, such as breast cancer (BC), and could be used as a drug target for therapy. However, the functions of the protein or the pathways through which it increases cell proliferation and invasiveness of cancer cells are not well-known. Therefore, we aim to investigate the functions of JTB by using in-solution digestion-based cellular proteomics of control and upregulated and downregulated JTB protein in MCF7 breast cancer cell line, taking account that in-solution digestion-based proteomics experiments are complementary to the initial in-gel based ones. Proteomics analysis allows investigation of protein dysregulation patterns that indicate the function of the protein and its interacting partners, as well as the pathways and biological processes through which it functions. We concluded that JTB dysregulation increases the epithelial-mesenchymal transition (EMT) potential and cell proliferation, harnessing cytoskeleton organization, apical junctional complex, metabolic reprogramming, and cellular proteostasis. Deregulated JTB expression was found to be associated with several proteins involved in mitochondrial organization and function, oxidative stress (OS), apoptosis, and interferon alpha and gamma signaling. Consistent and complementary to our previous results emerged by using in-gel based proteomics of transfected MCF7 cells, JTB-related proteins that are overexpressed in this experiment suggest the development of a more aggressive phenotype and behavior for this luminal type A non-invasive/poor-invasive human BC cell line that does not usually migrate or invade compared with the highly metastatic MDA-MB-231 cells. This more aggressive phenotype of MCF7 cells related to JTB dysregulation and detected by both in-gel and in-solution proteomics could be promoted by synergistic upregulation of EMT, Mitotic spindle and Fatty acid metabolism pathways. However, in both JTB dysregulated conditions, several downregulated JTB-interacting proteins predominantly sustain antitumor activities, attenuating some of the aggressive phenotypical and behavioral traits promoted by the overexpressed JTB-related partners.

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein-protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide "molecular pictures", which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.

Force-Induced Near-Infrared Chromism of Mechanophore-Linked Polymers.

A near-infrared (NIR) mechanophore was developed and incorporated into a poly(methyl acrylate) chain to showcase the first force-induced NIR chromism in polymeric materials. This mechanophore, based on benzo[1,3]oxazine (OX) fused with a heptamethine cyanine moiety, exhibited NIR mechanochromism in solution, thin-film, and bulk states. The mechanochemical activity was validated using UV-vis-NIR absorption/fluorescence spectroscopies, gel permeation chromatography (GPC), NMR, and DFT simulations. Our work demonstrates that NIR mechanochromic polymers have considerable potential in mechanical force sensing, damage detection, bioimaging, and biomechanics.

Proteomic Analysis of the Lake Trout (Salvelinus namaycush) Liver Identifies Proteins from Evolutionarily Close and -Distant Fish Relatives.

Lake trout are used as bioindicators for toxics exposure in the Great Lakes ecosystem. Here the first lake trout (Salvelinus namaycush) liver proteomics study is performed and searched against specific databases: (NCBI and UniProtKB) Salvelinus, Salmonidae, Actinopterygii, and Oncorhynchus mykiss, and the more distant relative, Danio rerio. In the biological replicate 1 (BR1), technical replicate 1 (TR1), (BR1TR1), a large number of lake trout liver proteins are not in the Salvelinus protein database, suggesting that lake trout liver proteins have homology to some proteins from the Salmonidae family and Actinopterygii class, and to Oncorhynchus mykiss and Danio rerio, two more highly studied fish. In the NCBI search, 4194 proteins are identified: 3069 proteins in Actinopterygii, 1617 in Salmonidae, 68 in Salvelinus, 568 in Oncorhynchus mykiss, and 946 in Danio rerio protein databases. Similar results are observed in the UniProtKB searches of BR1RT1, as well as in a technical replicate (BR1TR2), and then in a second biological replicate experiment, with two technical replicates (BR2TR1 and BR2TR2). This study opens the possibility of identifying evolutionary relationships (i.e., adaptive mutations) between various groups (i.e., zebrafish, rainbow trout, Salmonidae, Salvelinus and lake trout) through evolutionary proteomics. Data are available via the PRIDE Q2 (PXD011924).

Identification of dysregulation of atrial proteins in rats with chronic obstructive apnea using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two-dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta-oxidation, electron transport chain and Krebs cycle to be down-regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D-PAGE and nanoLC-MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down-regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D-PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181.

Investigation of Antibody-Drug Conjugates by Mass Spectrometry.

ADCs are empowered monoclonal antibodies that are designed to harness their targeting ability by linking them to cell-killing agents. They are made up of three main components, the antibody, linker and the cytotoxic drug. The specificity of the antibody with the antigen on the tumor cell surface helps with its internalization into the cell after which the active drug is released causing cell death. The investigation of ADCs can be done using a variety of MS methods. Here, we talk about the bottom-up approach, the top-down approaches such as ECD and ETD, the ESI/MS method and IM-MS. Further, we also focus on the applications of MALDI/MS such as UV-MALDI, IR-MALDI and IMS-MALDI and provide examples of the mass spectra that provide tremendous amount of information on ADC structures.

Correction to: 2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications.

This chapter was previously published as non-open access. This is updated to an open access chapter now.

Mass Spectrometry Based Comparative Proteomics Using One Dimensional and Two Dimensional SDS-PAGE of Rat Atria Induced with Obstructive Sleep Apnea.

Proteomics involves large-scale comprehensive study of specific proteomes which have been widely used in the field of biomarker discovery, drug development, disease diagnosis and therapy. Comprehensive proteomics involve two or more proteomics approaches that are confirmatory, complementary, and/or synergistic. Obstructive sleep apnea (OSA) is a sleep disorder which causes respiratory cessation (due to upper airway collapse). Here we describe a comprehensive MS based label-free quantitative proteomic analysis of the OSA induced rat atria homogenates and matched controls by using 1 dimensional SDS PAGE (1-D PAGE) and 2 dimensional SDS PAGE (2-D PAGE) separation of the proteins, enzymatic digestion and analysis by nanoliquid chromatography tandem-mass spectrometry (LC-MS/MS). The outcomes from the 1D-PAGE and 2D-PAGE studies not only identified dysregulated proteins due to OSA, but also confirmed and complemented each other.

2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications.

Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS PAGE) is a method that separates proteins according to their isoelectric points in the first dimension and molecular masses in the second dimension. Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when dissolved in a mixture of both buffers. Quantification of 60 proteins in rat liver cytosol over a wide range of pI and MW gave linear plots of spot density versus total protein for loads of 200, 400 and 600 µg protein dissolved in SDS buffer and run in triplicate on 2D gels (Average R2 = 0.987). Examples of biomedical applications are provided in which 2D proteins of interest found by comparing stained or western blotted 2D gel patterns were identified by mass spectrometry (MS).