Mining of serum glycoproteins by an indirect approach using cell line secretome
ABSTRACT Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins is challenging due to its wide dynamic range. Alternatively, glycoproteins can be discovered in the secretome of model cell lines and then confirmed in blood. However, there has been little experimental evidence showing cell line secretome as a tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS analysis. Among the identified proteins, we selected 13 proteins that had one or more N-glycosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma Proteome Database (PPD), and whose antibodies were commercially available. Nine out of the 13 selected proteins were detected from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS, which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant serum proteins.
- SourceAvailable from: Obi Lee Griffith[Show abstract] [Hide abstract]
ABSTRACT: We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.Journal of Proteome Research 02/2012; 11(4):2508-20. DOI:10.1021/pr201206w · 4.25 Impact Factor
Article: Sieving through the cancer secretome[Show abstract] [Hide abstract]
ABSTRACT: Cancer is among the most prevalent and serious health problems worldwide. Therefore, there is an urgent need for novel cancer biomarkers with high sensitivity and specificity for early detection and management of the disease. The cancer secretome, encompassing all the proteins that are secreted by cancer cells, is a promising source of biomarkers as the secreted proteins are most likely to enter the blood circulation. Moreover, since secreted proteins are responsible for signaling and communication with the tumor microenvironment, studying the cancer secretome would further the understanding of cancer biology. Latest developments in proteomics technologies have significantly advanced the study of the cancer secretome. In this review, we will present an overview of the secretome sample preparation process and summarize the data from recent secretome studies of six common cancers with high mortality (breast, colorectal, gastric, liver, lung and prostate cancers). In particular, we will focus on the various platforms that were employed and discuss the clinical applicability of the key findings in these studies. This article is part of a Special Issue entitled: An Updated Secretome.Biochimica et Biophysica Acta 01/2013; 1834(11). DOI:10.1016/j.bbapap.2013.01.030 · 4.66 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Despite major improvements on the knowledge and clinical management, cancer is still a deadly disease. Novel biomarkers for better cancer detection, diagnosis and treatment prediction are urgently needed. Proteins secreted, shed or leaking from the cancer cell, collectively termed the cancer secretome, are promising biomarkers since they might be detectable in blood or other biofluids. Furthermore, the cancer secretome in part represents the tumor microenvironment that plays a key role in tumor promoting processes such as angiogenesis and invasion. The cancer secretome, sampled as conditioned medium from cell lines, tumor/tissue interstitial fluid or tumor proximal body fluids, can be studied comprehensively by nanoLC-MS/MS-based approaches. Here, we outline the importance of current cancer secretome research and describe the mass spectrometry-based analysis of the secretome. Further, we provide an overview of cancer secretome research with a focus on the three most common cancer types: lung, breast and colorectal cancer. We conclude that the cancer secretome research field is a young, but rapidly evolving research field. Up to now, the focus has mainly been on the discovery of novel promising secreted cancer biomarker proteins. An interesting finding that merits attention is that in cancer unconventional secretion, e.g. via vesicles, seems increased. Refinement of current approaches and methods and progress in clinical validation of the current findings are vital in order to move towards applications in cancer management. This article is part of a Special Issue entitled: An Updated Secretome.Biochimica et Biophysica Acta 01/2013; 1834(11). DOI:10.1016/j.bbapap.2013.01.029 · 4.66 Impact Factor