Quantification of Protein Biomarkers of Preterm Birth by Stable Isotope Dilution LC-MS/MS Method

摘要

Preterm labor is defined as presence of uterine contractions before term gestation (between 20 and 37 weeks). Preterm labor precedes almost half of preterm births and is the leading contributor of perinatal morbidity and mortality. Early identification of at-risk women can alleviate this health issue, however, the etiology and mechanisms of preterm labor are not well understood and hence the need to identify novel biomarkers of this serious medical condition. Cervical-vaginal fluid (CVF) is a complex mixture of secretions from the vagina, endocervix, decidua and amniochorion; and can potentially serve as an important diagnostic site to monitor maternal and fetal health in pregnant women because of its minimally invasive collection method. The present study utilized stable isotope labeling with amino acids in cell culture (SILAC) to generate [~(13)C_(6) ~(15)N_(2)]-Lys, [~(13)C_(6)~(15)N_(1)]-Leu-labeled proteins secreted by immortalized human endocervical (End 1) and vaginal (VK2) cells in culture. SILAC-labeled End1 and VK2 supernatants were depleted twice of six major serum proteins by HPLC-based Multiple-Affinity-Removal-System (MARS), combined in 1:1 ratio (200 (mu)g/replicate, 3 replicates), and digested with trypsin. The tryptic peptides were fractionated by strong cation exchange chromatography into nine pooled fractions; each fraction further separated by microflow LC-coupled with an ESI-linear ion trap (LTQ) mass spectrometer and analyzed under full scan data dependent acquisition mode (LC-MS/MS). Peptides were identified against a human database using Sequest and Mascot search algorithms, and the data were refined for high confidence proteins using Peptide Prophet~R, Protein Prophet~R and Scaffold~R softwares. This methodology identified 445 unique proteins in the End1-Vk2-SILAC secretome to be used as Internal Standard for analyses of clinical samples. Ten proteins that were considered to be most relevant to preterm birth were short-listed, one unique peptide/protein was selected and MRMs were generated based on their full-scan LC-MS/MS data. For clinical samples, protein (40 (mu)g/patient) from either 5 term-pregnancy patients (CONTROL) or 5 preterm-pregnancy patients (PTB) were pooled together, mixed with 200 (mu)g of End1-Vk2 (1:1) SILAC-Internal Standard, processed as described above and estimated for the 10 proteins by LC-MRM/MS assays. This method provides for relative quantification of 10 proteins/analysis that are normalized with the SILAC-generated isotopically labeled proteins. In summary, this SILAC-based approach has generated an extremely rich pool of 445 isotopically labeled proteins that have been spiked as internal standards in human CVF samples to facilitate relative quantitation of CVF proteins, and thereby aid in developing a panel of novel biomarkers of preterm birth. Supported by NIH Grant P30ES013508.