Immunohistochemistry (IHC) is still widely used as a morphology-based assay for in situ analysis of target proteins as specific tumor antigens. However, as a very heterogeneous collection of neoplastic diseases, breast cancer (BC) requires an accurate identification and characterization of larger panels of candidate biomarkers, beyond ER, PR, and HER2 proteins, for diagnosis and personalized treatment, without the limited availability of antibodies that are required to identify specific proteins. Top-down, middle-down, and bottom-up mass spectrometry (MS)-based proteomics approaches complement traditional histopathological tissue analysis to examine expression, modification, and interaction of hundreds to thousands of proteins simultaneously. In this review, we discuss the proteomics-based identification of dysregulated proteins in BC that are essential for the following issues: discovery and validation of new biomarkers by analysis of solid and liquid/non-invasive biopsies, cell lines, organoids and xenograft models; identification of panels of biomarkers for early detection and accurate discrimination between cancer, benign and normal tissues; identification of subtype-specific and stage-specific protein expression profiles in BC grading and measurement of disease progression; characterization of new subtypes of BC; characterization and quantitation of post-translational modifications (PTMs) and aberrant protein–protein interactions (PPI) involved in tumor development; characterization of the global remodeling of BC tissue homeostasis, diagnosis and prognostic information; and deciphering of molecular functions, biological processes and mechanisms through which the dysregulated proteins cause tumor initiation, invasion, and treatment resistance.

It is thought that accurate risk assessment and early diagnosis of breast cancer (BC) can help reduce cancer-related mortality. Proteomics analysis of breast milk may provide biomarkers of risk and occult disease. Our group works on the analysis of human milk samples from women with BC and controls to investigate alterations in protein patterns of milk that could be related to BC. In the current study, we used mass spectrometry (MS)-based proteomics analysis of 12 milk samples from donors with BC and matched controls. Specifically, we used one-dimensional (1D)-polyacrylamide gel electrophoresis (PAGE) coupled with nanoliquid chromatography tandem MS (nanoLC-MS/MS), followed by bioinformatics analysis. We confirmed the dysregulation of several proteins identified previously in a different set of milk samples. We also identified additional dysregulations in milk proteins shown to play a role in cancer development, such as Lactadherin isoform A, O-linked N-acetylglucosamine (GlcNAc) transferase, galactosyltransferase, recoverin, perilipin-3 isoform 1, histone-lysine methyltransferase, or clathrin heavy chain. Our results expand our current understanding of using milk as a biological fluid for identification of BC-related dysregulated proteins. Overall, our results also indicate that milk has the potential to be used for BC biomarker discovery, early detection and risk assessment in young, reproductively active women.

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.

Invasive ductal carcinoma (IDC) is the most common histological subtype of malignant breast cancer (BC), and accounts for 70–80% of all invasive BCs. IDC demonstrates great heterogeneity in clinical and histopathological characteristics, prognoses, treatment strategies, gene expressions, and proteomic profiles. Significant proteomic determinants of the progression from intraductal pre-invasive malignant lesions of the breast, which characterize a ductal carcinoma in situ (DCIS), to IDC, are still poorly identified, validated, and clinically applied. In the era of “6P” medicine, it remains a great challenge to determine which patients should be over-treated versus which need to be actively monitored without aggressive treatment. The major difficulties for designating DCIS to IDC progression may be solved by understanding the integrated genomic, transcriptomic, and proteomic bases of invasion. In this review, we showed that multiple proteomics-based techniques, such as LC–MS/MS, MALDI-ToF MS, SELDI-ToF-MS, MALDI-ToF/ToF MS, MALDI-MSI or MasSpec Pen, applied to in-tissue, off-tissue, BC cell lines and liquid biopsies, improve the diagnosis of IDC, as well as its prognosis and treatment monitoring. Classic proteomics strategies that allow the identification of dysregulated protein expressions, biological processes, and interrelated pathway analyses based on aberrant protein–protein interaction (PPI) networks have been improved to perform non-invasive/minimally invasive biomarker detection of early-stage IDC. Thus, in modern surgical oncology, highly sensitive, rapid, and accurate MS-based detection has been coupled with “proteome point sampling” methods that allow for proteomic profiling by in vivo “proteome point characterization”, or by minimal tissue removal, for ex vivo accurate differentiation and delimitation of IDC. For the detection of low-molecular-weight proteins and protein fragments in bodily fluids, LC–MS/MS and MALDI-MS techniques may be coupled to enrich and capture methods which allow for the identification of early-stage IDC protein biomarkers that were previously invisible for MS-based techniques. Moreover, the detection and characterization of protein isoforms, including posttranslational modifications of proteins (PTMs), is also essential to emphasize specific molecular mechanisms, and to assure the early-stage detection of IDC of the breast.

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.

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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.

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.