Subunit-specific mass spectrometry method identifies haptoglobin subunit alpha as a diagnostic marker in non-small cell lung cancer.

Haptoglobin (Hp) subunits have been suggested as a potential serum marker for lung cancer. Research is intense on the application of Hp subunits to predict the cancer earlier. Nevertheless, it remains difficult to accurately measure the content of Hp subunits. We developed stable isotope dilution-multiple reaction monitoring mass spectrometry (SID-MRM-MS) capable of measuring Hp subunits (alpha and beta chains). Three isotopic analogs (NPANPVQ, TEGDGVYTLNDK and ILGGHLDAK for alpha, alpha2 and beta chain, respectively) were used as internal standard (IS) for SID-MRM-MS. Serum levels of each Hp subunit were measured in 210 clinical samples using SID-MRM-MS. A concentration ratio of each Hp subunit to total Hp was investigated. Secretion levels of alpha and beta chains were significantly increased in non-small cell lung cancer (NSCLC) compared to controls (P<0.0001). Alterations of the alpha chain ratio were more apparent than beta chain between controls and NSCLC (P=0.0001 and 0.338 for alpha and beta chains, respectively). In conclusion, this study provides not only an efficient quantitative method to determine each Hp subunit in crude sera, but also evidence that Hp alpha chain is a more prospective biomarker to diagnose NSCLC than beta chain. BIOLOGICAL SIGNIFICANCE: Recent several studies have reported Hp as a potential biomarker for diagnosis of lung cancer. However a successful evaluation of the value of Hp subunits was not achieved on clinical samples. To evaluate the diagnostic performance of each Hp subunit, the development of an accurate quantitative assay of Hp subunits is necessary. In this regard, we employed a new analytical method using stable isotope dilution-multiple reaction monitoring mass spectrometry (SID-MRM-MS), capable of measuring Hp subunits in 210 clinical specimens. In this article, we measured the Hp subunit concentrations and Hp subunits/total Hp ratios in patients with NSCLC using SID-MRM-MS. This is the first report on the evaluation of each Hp subunit as a lung cancer marker using SID-MRM-MS. Consequently, we evaluated specific three tryptic peptides (e.g. NPANPVQ, TEGDGVYTLNDK and ILGGHLDAK for alpha, alpha2 and beta chain, respectively) with high specificity and sensitivity for determination of Hp subunits. Through future large prospective cohort studies, the clinical application of Hp subunits as complementary markers, especially Hp alpha, would be useful for the diagnosis of NSCLC.