title: Subunit-specific mass spectrometry method identifies haptoglobin subunit alpha as a diagnostic marker in non-small cell lung cancer.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, South Korea.
authors.forename: Jisook
authors.initials: J
authors.lastname: Park
authors.forename: Jeong Soo
authors.initials: JS
authors.lastname: Yang
authors.forename: Gyuyon
authors.initials: G
authors.lastname: Jung
authors.forename: Hye In
authors.initials: HI
authors.lastname: Woo
authors.forename: Hyung-Doo
authors.initials: HD
authors.lastname: Park
authors.forename: Jong-Won
authors.initials: JW
authors.lastname: Kim
authors.forename: Wooseong
authors.initials: W
authors.lastname: Huh
authors.forename: Jae-Wook
authors.initials: JW
authors.lastname: Ko
authors.forename: Hojoong
authors.initials: H
authors.lastname: Kim
authors.forename: Je-Yoel
authors.initials: JY
authors.lastname: Cho
authors.forename: Soo-Youn
authors.initials: SY
authors.lastname: Lee
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood*; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood* / Q000473:pathology; D006242:Haptoglobins / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood* / Q000473:pathology; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000097:blood*
chemical_list: D014408:Biomarkers, Tumor; C485359:HP protein, human; D006242:Haptoglobins; D009363:Neoplasm Proteins
keywords: AC; AUC; Biomarker; CEA; CYFRA 21-1; DP; ELISA; EPI; Haptoglobin; Hp; IS; Lung cancer; Mass spectrometry; Multiple reaction monitoring; NSCLC; NSE; PTM; ProGRP; ROC; SCC; SCLC; SID–MRM-MS; adenocarcinoma; area under the ROC curve; carcinoembryonic antigen; cytokeratin fragment 19; declustering potential; enhanced product ion; enzyme-linked immunosorbent assay; haptoglobin; internal standard; neuron specific enolase; non-small cell lung cancer; post-translational modification; pro-gastrin-releasing peptide; receiver operating characteristic; small cell lung cancer; squamous cell carcinoma; stable isotope dilution–multiple reaction monitoring mass spectrometry