Integrated Pipeline for Mass Spectrometry-Based Discovery and Confirmation of Biomarkers Demonstrated in a Mouse Model of Breast Cancer

Abstract

Despite their potential to impact diagnosis and treatment of cancer, few protein biomarkers are in clinical use. Biomarker discovery is plagued with difficulties ranging from technological (inability to globally interrogate proteomes) to biological (genetic and environmental differences among patients and their tumors). We urgently need paradigms for biomarker discovery. To minimize biological variation and facilitate testing of proteomic approaches, we employed a mouse model of breast cancer. Specifically, we performed LC-MS/MS of tumor and normal mammary tissue from a conditional HER2/Neu-driven mouse model of breast cancer, identifying 6758 peptides representing >700 proteins. We developed a novel statistical approach (SASPECT) for prioritizing proteins differentially represented in LC-MS/MS datasets and identified proteins over- or under-represented in tumors. Using a combination of antibody-based approaches and multiple reaction monitoring-mass spectrometry (MRM-MS), we confirmed the overproduction of multiple proteins at the tissue level, identified fibulin-2 as a plasma biomarker, and extensively characterized osteopontin as a plasma biomarker capable of early disease detection in the mouse. Our results show that a staged pipeline employing shotgun-based comparative proteomics for biomarker discovery and multiple reaction monitoring for confirmation of biomarker candidates is capable of finding novel tissue and plasma biomarkers in a mouse model of breast cancer. Furthermore, the approach can be extended to find biomarkers relevant to human disease.

Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

“…This is an area where MRM-MS is really gaining interest and momentum. Not only can a targeted approach such as MRM-MS lessen the ever challenging dynamic range issues most commonly encountered during global proteomics experiments thereby digging deeper into the low abundance proteome and low stochiometric PTM, this methodology is showing tremendous utility in the verification of both global proteomics identification and quantification data [12,23,[35][36][37][38][39]. Unfortunately, global proteomics data can be inconclusive.…”

“…This method was further optimized for greater precision and accuracy by Whiteaker et al, reporting a physiologically relevant sensitivity in the ng/mL range [50]. Whiteaker et al also utilized the a combination of antibody and MS approaches to confirm global proteomics results from mouse model of breast cancer while characterizing and transferring a couple of the proteins identified previously to a plasma biomarker [38]. Finally, Kornilayev et al, extended the SISCAPA approach developed by Anderson et al by utilizing polyclonal monospecific anti-peptide antibodies raised against unique cytochrome P450 isoenzymes specific tryptic peptides for qualitative and quantitative proteomic analysis.…”

“…Growing interest in using this technology to quantify proteins has led to a number of new software developments over the last few years. As state above, the MIDAS and TIQAM workflows were developed for targeted detection of proteins [21,44], their respective PTM [8,41] and for the rapid development of MRM assays [1,23,53]. Informatic strategies for the prediction of the most likely peptides (proteotypic peptides) that will be observed for proteins of interest are emerging [14,15,23,54,55].…”

Setup for human sera MALDI profiling: The case of rhEPO treatment

“…Despite the strong potential of MALDI, the main issue to face remains the serum dynamic range (i.e. the range of serum protein concentrations from high-to low-abundance proteins, estimated in ten orders of magnitude [11]) which encompasses eight orders of magnitude the capacity of the mass spectrometer to discriminate species in complex mixtures [12][13][14][15] preventing the detection of biomarkers from picogram to femtogram levels, such as prostate specific antigen (PSA) or interleukins, leaving undisclosed a large number of proteins and peptides. So far, to overcome this issue, an adequate fractionation step prior proteomic analysis is mandatory in order to remove high-abundant components.…”

Development of a high-throughput IMS–IMS–MS approach for analyzing mixtures of biomolecules

“…Methods include the dynamic range enhancement applied to mass spectrometry (DREAMS) approach which is used with Fourier Transform MS and multiple reaction monitoring (MRM) used extensively in tandem MS instrumentation. [14][15][16][17][18][19] We have demonstrated a different approach that combines a gas-phase separation, ion mobility spectrometry (IMS), 20-25 with time-of-flight (TOF) MS. 26,27 Here, the initial separation of ions according to their mobilities reduces spectral congestion associated with chemical noise. 28 Thus incorporation of an IMS separation step prior to MS analysis provides a means of increasing coverage and sensitivity while maintaining high analysis speeds.…”