Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS)-Based Targeted Proteomic Surrogates for Intrinsic Subtypes in Breast Cancer: Comparative Analysis with Immunohistochemical Phenotypes

Abstract

Advances in targeted medications have improved the survival rate of breast cancer patients with molecular marker-positive tumors. To date, immunohistochemistry (IHC) has remained as the standard method for quantifying the markers including HER2, ER, and PR. Nevertheless, IHC-based grading is subjective, because the results depend on trained individuals’ eye rather than numerical quantities. Thus, alternative methods that can account for quantitative levels of markers are gaining popularity, including targeted proteomics by mass spectrometry (MS). However, technical limitations have impeded the application of MS-based protein quantification to pathological FFPE slides that contain low amounts of cross-linked proteins. To challenge this, we developed a parallel reaction monitoring-mass spectrometry (PRM-MS) method to measure the expression levels of breast cancer markers. After developing the method using cell lines, we performed PRM-MS using 51 individuals’ FFPE samples. As a result, we obtained numerical measures of targets, quantifying 13 peptides of 4 markers in a single analysis per sample. The results correlated well with the IHC readings of experienced pathologists. Moreover, the results distinguished a gray zone in HER2 classification, which IHC alone failed to do. This proof-of-concept study demonstrates the application of targeted proteomics in pathologic slides, further supporting the applicability of MS-based approaches in precision medicine.

Transcriptome profiling and proteomic validation reveals targets of the androgen receptor signaling in the BT-474 breast cancer cell line

“…The novel targeted MS-based approach of heavy-peptide labelled PRM shows high analytical performance, high sensitivity and high mass accuracy [ 78 , 79 ]. This may be a useful tool for further implementation in more relevant systems [ 80 ], such as in cell line in vivo models, and patient-derived xenograft models [ 81 , 82 ], with potential to be ultimately used in clinical assays. Thus, precision medicine can benefit from this approach by identifying biomarkers for monitoring disease progression or response to treatment, based on the patient’s proteome [ 81 , 82 ].…”

Proteomic Analysis Reveals a Novel Therapeutic Strategy Using Fludarabine for Steroid-Resistant Asthma Exacerbation

“…Parallel reaction monitoring (PRM) proteomic is an ion monitoring technique based on high-resolution mass spectrometry which is more convenient in assay development for precise quantification of proteins and peptides of interest (15). Among all major protein components for the immunopathogenesis of asthma exacerbation, using label-free shotgun proteomic approach, STAT1 was found to be among the most highly relevant protein and was then validated by PRM mass spectrometry.…”

Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

“…cerebrospinal fluid [131], urine [132][133][134], and formalin-fixed paraffinembedded (FFPE) samples [135][136][137]. These analyses have sought to quantify the protein biomarkers of breast cancer [21,[137][138][139][140], colorectal cancer [141][142][143][144][145], lung cancer [146][147][148][149], oral cancer [150,151], multiple sclerosis [131,152], cardiovascular diseases [153][154][155][156][157][158],…”