Proteome Profiling of Breast Tumors by Gel Electrophoresis and Nanoscale Electrospray Ionization Mass Spectrometry

Abstract

We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future.

Comparative proteomic analysis of ductal and lobular invasive breast carcinoma

“…We also confirmed the increased expression of the PDIA3 gene in IDC compared with normal breast tissue (P < 0.001) by RT-qPCR and found differential expression of both genes in more aggressive tumor subsets, such as in lymph node metastasis and grade III tumors (Ramos et al, 2015). A comparative proteomic study of breast tumors and adjacent normal tissue also showed increased expression of PDIA3 and PDIA6 proteins in breast carcinoma (Alldridge et al, 2008). Pressinotti et al (2009) investigated the differential expression of PDIA3 in prostate cancers.…”

The Expressions of Cancer Stem Cell Markers and Nonclassical HLA antigens in Breast Tumors

“…It has been reported that Nanog expression up-regulates most cancer types such as carcinoma in situ, embryonal carcinomas, and cervical and ovarian cancers (39). Mass spectrophotometric analysis of frozen BC specimens showed that Nanog is one of the most highly increased proteins (40). Oct4, similar to Nanog, is an essential transcription factor for maintaining the stemness of both ESCs and CSCs (41)(42)(43).…”

Quantitative proteome profiling of lymph node-positivevs. -negative colorectal carcinomas pinpoints MX1 as a marker for lymph node metastasis

“…The proteins were extracted from the frozen tumor samples with 2X SDS sample buffer, reduced, alkylated and separated by gel electrophoresis as previously described 5 . The gel lanes were sliced and digested as described previously 5 .…”