Lung cancer, with its high metastatic potential and high mortality rate, is the worldwide leading cause of cancer-related deaths. High-throughput "omics"-based platforms have accelerated the discovery of biomarkers for lung cancer, and the resulting candidates are to be evaluated for their diagnostic potential as noninvasive biomarkers. The evaluation of the biomarker candidates involves the quantitative measurement of large numbers of proteins in bodily fluids using advanced mass spectrometric techniques. In this study, a robust pipeline based on targeted proteomics was developed for biomarker verification in plasma samples and applied to verifying lung cancer biomarker candidates. Highly multiplexed liquid chromatrography-selected reaction monitoring (LC-SRM) assays for 95 potential tumor markers for non-small-cell lung cancer (NSCLC) were generated to screen plasma samples obtained from 72, early to late stage, patients. A total of 17 proteins were verified as potent tumor markers detectable in plasma and, where available, verified by enzyme-linked immunosorbent assays (ELISAs). A novel plasma-based biomarker, zyxin, fulfilled the criteria for a potential early diagnostic marker for NSCLC.

Apelin peptides were recently identified as endogenous ligands of the APJ receptor. It has been hypothesized that these peptides are initially provided to the newborn by nursing and might be involved in gastrointestinal tract development. As apelin peptides may have different effects on the APJ receptor as a function of their size, knowledge of their exact structure in early milk is essential to clarify their action in gastrointestinal tract development. Bovine colostrum is thought to contain high concentrations of a wide diversity of apelin peptides, but none of them have yet been rigorously characterized. To identify and monitor apelin peptides in bovine colostrum, we developed a cation exchange extraction step followed by untargeted liquid chromatography coupled to high resolution and high mass accuracy mass spectrometry (LTQ-Orbitrap). Using this approach, we characterized 46 endogenous apelin peptides in bovine colostrum, which varied in relative abundance from one colostrum to another. Mature as well as commercial milk samples were also studied. Taken together, our data demonstrate that the multiplicity and variability of apelin peptides are biologically relevant and change during milk maturation to reach a more constant composition in mature milk.

Apelin peptides are of great interest owing to their involvement in physiological and pathological processes and they have been proposed as novel biomarkers for heart failure. The plasma concentrations of bioactive peptides of 12 (apelin-12), 13 (apelin-13) and pyroglutamyl apelin-13 (apelin-p13), 17 (apelin-17) and 36 (apelin-36) amino acids are reported to range from 20 to 4000 pg/mL in healthy subjects. As standard immunoassays cannot specifically quantify each apelin peptide, we have developed a sensitive and targeted multiplexed liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for each plasma apelin fragment. The approach was based on a cation-exchange extraction step of apelin forms present in human plasma. Apelin-12, -13, -p13, -17 and -36 were quantified using a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode. Stable isotope-labeled internal standards were used for quantification. Following assay validation, apelin peptide stability in plasma was investigated. Ten plasma samples from healthy donors were analyzed both with a standard immunoassay and with our LC/MS/MS method. The immunoassay results for the ten healthy donors showed immunoreactive plasma apelin concentrations ranging from 208 to 466 pg/mL. The lower limits of detection of our LC/MS/MS assay ranged from 10 to 50 pg/mL for apelin-12, -13, -p13, -17, and -36. Surprisingly, none of the five expected circulating forms of apelin was detected. These results question the nature and/or the concentration of circulating apelin peptides as well as the specificity of the immunoassays that have hitherto been used for clinical applications.