The utility of isotope-coded protein labeling for prioritization of proteins found in ovarian cancer patient urine

Abstract

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.

Discovery and Validation of Novel Protein Biomarkers in Ovarian Cancer Patient Urine

“…Validation of protein abundance differences using antibody‐based approaches is problematic . Western blotting was unable to identify or validate protein changes, due to a lack of specificity and the detection of multiple reactive bands in urine samples.…”

“…Only a single study has applied LFQ to examine the urine peptidome of ovarian cancer patients versus healthy controls . In previous work, we used isotope‐coded protein labeling (ICPL) and MALDI MS/MS to identify urinary biomarkers of ovarian cancer . Whilst several potential disease biomarkers—both established and novel—were identified, both the dynamic range of the ICPL‐MALDI approach and our ability to accurately quantitate changes was limited …”

“…[18] In previous work, we used isotope-coded protein labeling (ICPL) and MALDI MS/MS to identify urinary biomarkers of ovarian cancer. [19] Whilst several potential disease biomarkers-both established and novel-were identified, both the dynamic range of the ICPL-MALDI approach and our ability to accurately quantitate changes was limited. [19] Here we report a pilot study applying LFQ, followed by targeted parallel reaction monitoring (PRM) to discover and validate ovarian cancer-related protein changes in urine samples from multiple individual patients.…”

“…[19] Whilst several potential disease biomarkers-both established and novel-were identified, both the dynamic range of the ICPL-MALDI approach and our ability to accurately quantitate changes was limited. [19] Here we report a pilot study applying LFQ, followed by targeted parallel reaction monitoring (PRM) to discover and validate ovarian cancer-related protein changes in urine samples from multiple individual patients. Discovery experiments using LFQ were conducted in a cohort of patients (n = 20; cohort 1), and were followed by PRM validation using an independent patient cohort (n = 20; cohort 2).…”

Accurate quantification of modified cyclic peptides without the need for authentic standards

“…11,12 For quantification purposes, it is necessary to address these issues in particular ionisation efficiency and matrix effects when using an ESMS or MALDI-MS direct measurements. For this reason various sample treatments for MSbased quantification are reported in the literature for peptides including; isotope-coded affinity tag reagents (ICATs), [13][14][15] isotope-coded protein labelling (ICPL), [16][17][18] stable isotope labelling by amino acid in cell culture (SILAC), [19][20][21] isotopedifferentiated binding energy shift tag (IDBEST), chemical labelling, isobaric tagging (iTRAQ, TMT), 22,23 and absolute quantification with the use of synthetic labelled peptides (AQUA), 24,25 These methods require additional sample preparation and cost.…”