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1.  The Development of an Integrated Platform to Identify Breast Cancer Glycoproteome Changes in Human Serum 
Journal of chromatography. A  2009;1217(19):3307-3315.
Protein glycosylation represents one of the major post translational modifications and can have significant effects on protein function. Moreover, changes in the carbohydrate structure are increasingly being recognized as an important modification associated with cancer etiology. In this report, we describe the development of a proteomics approach to identify breast cancer related changes in either concentration and/or the carbohydrate structures of glycoprotein(s) present in blood samples. Diseased and healthy serum samples were processed by an optimized sample preparation protocol using multiple lectin affinity chromatography (M-LAC) that partitions serum proteins based on glycan characteristics. Subsequently, three separate procedures, 1D SDS-PAGE, isoelectric focusing and an antibody microarray, were applied to identify potential candidate markers for future study. The combination of these three platforms is illustrated in this report with the analysis of control and cancer glycoproteomic fractions. Firstly, a molecular weight based separation of glycoproteins by 1D SDS-PAGE was performed, followed by protein, glycoprotein staining, lectin blotting and LC-MS analysis. To refine or confirm the list of interesting glycoproteins, isoelectric focusing (targeting sialic acid changes) and an antibody microarray (used to detect neutral glycan shifts) were selected as the orthogonal methods. As a result, several glycoproteins including alpha-1B-glycoprotein, complement C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study.
PMCID: PMC4142217  PMID: 19782370
High performance multi-lectin affinity chromatography; lectin blotting; isoelectric focusing; lectin – antibody microarray
2.  Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins 
Nature methods  2013;11(2):149-155.
The successful application of MRM in biological specimens raises the exciting possibility that assays can be configured to measure all human proteins, resulting in an assay resource that would promote advances in biomedical research. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort in MRM assay generation. We have configured, validated across three laboratories, and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analyte in a panel of breast cancer-related cell lines. Median assay precision was 5.4%, with high inter-laboratory correlation (R2 >0.96). Peptide measurements in breast cancer cell lines were able to discriminate amongst molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a scaled, international effort.
PMCID: PMC3922286  PMID: 24317253
4.  Multiplexed quantification of estrogen receptor and HER2/Neu in tissue and cell lysates by peptide immunoaffinity enrichment mass spectrometry 
Proteomics  2012;12(8):1253-1260.
Access to a wider range of quantitative protein assays would significantly impact the number and use of tissue markers in guiding disease treatment. Quantitative mass spectrometry-based peptide and protein assays, such as immuno-SRM assays, have seen tremendous growth in recent years in application to protein quantification in biological fluids such as plasma or urine. Here, we extend the capability of the technique by demonstrating the application of a multiplexed immuno-SRM assay for quantification of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) levels in cell line lysates and human surgical specimens. The performance of the assay was characterized using peptide response curves, with linear ranges covering approximately 4 orders of magnitude and limits of detection in the low fmol/mg lysate range. Reproducibility was acceptable with median coefficients of variation of approximately 10%. We applied the assay to measurements of ER and HER2 in well-characterized cell line lysates with good discernment based on ER/HER2 status. Finally, the proteins were measured in surgically resected breast cancers, and the results showed good correlation with ER/HER2 status determined by clinical assays. This is the first implementation of the peptide-based immuno-SRM assay technology in cell lysates and human surgical specimens.
PMCID: PMC3418804  PMID: 22577026
Estrogen receptor; HER2/Neu; immunoaffinity; peptides; tissue
5.  Tumor Microenvironment-Derived Proteins Dominate the Plasma Proteome Response During Breast Cancer Induction and Progression 
Cancer research  2011;71(15):5090-5100.
Tumor development relies upon essential contributions from the tumor microenvironment and host immune alterations. These contributions may inform the plasma proteome in a manner that could be exploited for cancer diagnosis and prognosis. In this study, we employed a systems biology approach to characterize the plasma proteome response in the inducible HER2/neu mouse model of breast cancer during tumor induction, progression and regression. Mass spectrometry data derived from ∼ 1.6 million spectra identified protein networks involved in wound healing, microenvironment and metabolism that coordinately changed during tumor development. The observed alterations developed prior to cancer detection, increased progressively with tumor growth, and reverted toward baseline with tumor regression. Gene expression and immunohistochemical analyses suggested that the cancer-associated plasma proteome was derived from transcriptional responses in the non-cancerous host tissues as well as the developing tumor. The proteomic signature was distinct from a non-specific response to inflammation. Overall, the developing tumor simultaneously engaged a number of innate physiological processes, including wound repair, immune response, coagulation and complement cascades, tissue remodeling and metabolic homeostasis that were all detectable in plasma. Our findings offer an integrated view of tumor development with relevance to plasma-based strategies to detect and diagnose cancer.
PMCID: PMC3148311  PMID: 21653680
6.  A targeted proteomics–based pipeline for verification of biomarkers in plasma 
Nature biotechnology  2011;29(7):625-634.
High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.
PMCID: PMC3232032  PMID: 21685906
7.  Detection of Elevated Plasma Levels of EGF Receptor Prior to Breast Cancer Diagnosis among Hormone Therapy Users 
Cancer research  2010;70(21):8598-8606.
Applying advanced proteomic technologies to prospectively collected specimens from large studies is one means of identifying preclinical changes in plasma proteins that are potentially relevant to the early detection of diseases like breast cancer. We conducted fourteen independent quantitative proteomics experiments comparing pooled plasma samples collected from 420 estrogen receptor positive (ER+) breast cancer patients ≤17 months prior to their diagnosis and matched controls. Based on the over 3.4 million tandem mass spectra collected in the discovery set, 503 proteins were quantified of which 57 differentiated cases from controls with a p-value<0.1. Seven of these proteins, for which quantitative ELISA assays were available, were assessed in an independent validation set. Of these candidates, epidermal growth factor receptor (EGFR) was validated as a predictor of breast cancer risk in an independent set of preclinical plasma samples for women overall [odds ratio (OR)=1.44, p-value=0.0008], and particularly for current users of estrogen plus progestin (E+P) menopausal hormone therapy (OR=2.49, p-value=0.0001). Among current E+P users EGFR's sensitivity for breast cancer risk was 31% with 90% specificity. While EGFR's sensitivity and specificity are insufficient for a clinically useful early detection biomarker, this study suggests that proteins that are elevated preclinically in women who go on to develop breast cancer can be discovered and validated using current proteomic technologies. Further studies are warranted to both examine the role of EGFR and to discover and validate other proteins that could potentially be used for breast cancer early detection.
PMCID: PMC2970770  PMID: 20959476
Breast cancer; epidermal growth factor receptor; menopausal hormone therapy
8.  Plasma Proteome Profiles Associated with Inflammation, Angiogenesis, and Cancer 
PLoS ONE  2011;6(5):e19721.
Tumor development is accompanied by a complex host systemic response, which includes inflammatory and angiogenic reactions. Both tumor-derived and systemic response proteins are detected in plasma from cancer patients. However, given their non-specific nature, systemic response proteins can confound the detection or diagnosis of neoplasia. Here, we have applied an in-depth quantitative proteomic approach to analyze plasma protein changes in mouse models of subacute irritant-driven inflammation, autoreactive inflammation, and matrix associated angiogenesis and compared results to previously described findings from mouse models of polyoma middle T-driven breast cancer and Pdx1-Cre KrasG12D Ink4a/Arf lox/lox -induced pancreatic cancer. Among the confounding models, approximately 1/3 of all quantified plasma proteins exhibited a significant change in abundance compared to control mice. Of the proteins that changed in abundance, the majority were unique to each model. Altered proteins included those involved in acute phase response, inflammation, extracellular matrix remodeling, angiogenesis, and TGFβ signaling. Comparison of changes in plasma proteins between the confounder models and the two cancer models revealed proteins that were restricted to the cancer-bearing mice, reflecting the known biology of these tumors. This approach provides a basis for distinguishing between protein changes in plasma that are cancer-related and those that are part of a non-specific host response.
PMCID: PMC3093388  PMID: 21589862
9.  The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans 
The Journal of Experimental Medicine  2009;206(7):1495-1503.
Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity.
PMCID: PMC2715080  PMID: 19528259
10.  Application of serum proteomics to the Women's Health Initiative conjugated equine estrogens trial reveals a multitude of effects relevant to clinical findings 
Genome Medicine  2009;1(4):47.
The availability of serum collections from the Women's Health Initiative (WHI) conjugated equine estrogens (CEE) randomized controlled trial provides an opportunity to test the potential of in-depth quantitative proteomics to uncover changes in the serum proteome related to CEE and to assess their relevance to trial findings, including elevations in the risk of stroke and venous thromboembolism and a reduction in fractures.
Five independent large scale quantitative proteomics analyses were performed, each comparing a set of pooled serum samples collected from 10 subjects, 1 year following initiation of CEE at 0.625 mg/d, relative to their baseline pool. A subset of proteins that exhibited increased levels with CEE by quantitative proteomics was selected for validation studies.
Of 611 proteins quantified based on differential stable isotope labeling, the levels of 116 (19%) were changed after 1 year of CEE (nominal P < 0.05), while 64 of these had estimated false discovery rates <0.05. Most of the changed proteins were not previously known to be affected by CEE and had relevance to processes that included coagulation, metabolism, osteogenesis, inflammation, and blood pressure maintenance. To validate quantitative proteomic data, 14 proteins were selected for ELISA. Findings for ten - IGF1, IGFBP4, IGFBP1, IGFBP2, F10, AHSG, GC, CP, MMP2, and PROZ - were confirmed in the initial set of 50 subjects and further validated in an independent set of 50 additional subjects who received CEE.
CEE affected a substantial fraction of the serum proteome, including proteins with relevance to findings from the WHI CEE trial related to cardiovascular disease and fracture.
Clinical Trials Registration identifier: NCT00000611
PMCID: PMC2684668  PMID: 19402886

Results 1-10 (10)