PMC:3431878 / 20503-25124 JSONTXT

{"project":"2_test","denotations":[{"id":"22949880-10228899-50810738","span":{"begin":416,"end":418},"obj":"10228899"},{"id":"22949880-12615972-50810739","span":{"begin":615,"end":617},"obj":"12615972"},{"id":"22949880-10228899-50810740","span":{"begin":618,"end":620},"obj":"10228899"},{"id":"22949880-11125279-50810741","span":{"begin":621,"end":623},"obj":"11125279"},{"id":"22949880-18334732-50810742","span":{"begin":624,"end":626},"obj":"18334732"},{"id":"22949880-21901274-50810743","span":{"begin":627,"end":629},"obj":"21901274"},{"id":"22949880-11369781-50810744","span":{"begin":1246,"end":1248},"obj":"11369781"},{"id":"22949880-11786729-50810745","span":{"begin":1249,"end":1251},"obj":"11786729"},{"id":"22949880-12218296-50810746","span":{"begin":1252,"end":1254},"obj":"12218296"},{"id":"22949880-12615972-50810747","span":{"begin":1656,"end":1658},"obj":"12615972"},{"id":"22949880-15905287-50810748","span":{"begin":1909,"end":1911},"obj":"15905287"},{"id":"22949880-12734200-50810749","span":{"begin":2209,"end":2210},"obj":"12734200"},{"id":"22949880-16901338-50810750","span":{"begin":2503,"end":2505},"obj":"16901338"},{"id":"22949880-17220477-50810751","span":{"begin":2960,"end":2962},"obj":"17220477"},{"id":"22949880-17604678-50810752","span":{"begin":3083,"end":3085},"obj":"17604678"},{"id":"22949880-17687369-50810753","span":{"begin":3567,"end":3569},"obj":"17687369"},{"id":"22949880-18688232-50810753","span":{"begin":3567,"end":3569},"obj":"18688232"},{"id":"22949880-18688233-50810753","span":{"begin":3567,"end":3569},"obj":"18688233"},{"id":"22949880-16525410-50810754","span":{"begin":4129,"end":4131},"obj":"16525410"},{"id":"22949880-21818808-50810755","span":{"begin":4494,"end":4496},"obj":"21818808"},{"id":"T46059","span":{"begin":416,"end":418},"obj":"10228899"},{"id":"T46141","span":{"begin":615,"end":617},"obj":"12615972"},{"id":"T35504","span":{"begin":618,"end":620},"obj":"10228899"},{"id":"T29950","span":{"begin":621,"end":623},"obj":"11125279"},{"id":"T91999","span":{"begin":624,"end":626},"obj":"18334732"},{"id":"T58996","span":{"begin":627,"end":629},"obj":"21901274"},{"id":"T29033","span":{"begin":1246,"end":1248},"obj":"11369781"},{"id":"T39769","span":{"begin":1249,"end":1251},"obj":"11786729"},{"id":"T53745","span":{"begin":1252,"end":1254},"obj":"12218296"},{"id":"T76798","span":{"begin":1656,"end":1658},"obj":"12615972"},{"id":"T13540","span":{"begin":1909,"end":1911},"obj":"15905287"},{"id":"T32166","span":{"begin":2209,"end":2210},"obj":"12734200"},{"id":"T71794","span":{"begin":2503,"end":2505},"obj":"16901338"},{"id":"T81494","span":{"begin":2960,"end":2962},"obj":"17220477"},{"id":"T98238","span":{"begin":3083,"end":3085},"obj":"17604678"},{"id":"T19354","span":{"begin":3567,"end":3569},"obj":"17687369"},{"id":"T84523","span":{"begin":3567,"end":3569},"obj":"18688232"},{"id":"T15186","span":{"begin":3567,"end":3569},"obj":"18688233"},{"id":"T57504","span":{"begin":4129,"end":4131},"obj":"16525410"},{"id":"T26089","span":{"begin":4494,"end":4496},"obj":"21818808"}],"text":"7. Mass Spectrometric Identification of CA125\nIsolation of CA125 for the analysis of its molecular features represents a challenge for researchers, as routine high resolution methods such as SDS-PAGE are not ideal for very high molecular weight analytes. Typically, size exclusion chromatography (SEC) is used for CA125 purification; however the separation efficiency is limited and results in cross-contaminations [16]. Additional methods used for the prefractionation of CA125 include immuno-precipitation, affinity chromatography or a combination of these methods that are associated with similar disadvantages [12,16,28,48,50]. Subsequent identification of CA125 in the derived fractions is therefore necessary for reliable downstream analysis. However, CA125 identification solely based on antibody probing alone can lead to false-positive protein assignment as a result of antibody cross-reactivity [41]. Thus, characterization of these secondary proteins may lead to erroneous assignment of molecular attributes to CA125. Several studies show that mass spectrometry (MS) offers a suitable solution for independent CA125 identification (discussed below). It is, however, notable that the published protein sequence for CA125 is incomplete [17,18,21]. Although this may result in some peptide identifications being missed by search algorithms, mass spectrometric identification of CA125 is a viable approach that can complement antibody based identification methods.\nThe first peptide mass fingerprinting data on CA125 was described in 2003 where 16 masses, corresponding to peptides of a 1148 amino acid form of CA125 from HeLa cells, were detected [12]. This enabled the identification of CA125 as a binding protein of galectin-1. In a later study, Jankovic et al. identified an approximately 200 kDa form of CA125 in first trimester human placental extract based on 36 experimentally detected masses [51]. Similarly, Milutinovic et al. reported identification of 46 masses that matched to CA125 in human amniotic fluid [52].\nTandem mass spectrometric data on CA125 was first reported by Kui Wong et al., confirming the identity of CA125 in a purified sample prior to oligosaccharide characterization [7]. Although only a single peptide was reported, this identification was supported by peptide mass fingerprinting where 11 experimentally derived masses were matched to theoretical peptides. Analyses of the tear proteome by tandem MS lead to the identification of 491 proteins including CA125 [53]. This was the first mass spectrometric data of CA125 that included statistical analysis and associated metadata for eight peptides of this protein. However, the molecular weight range of the fraction in which CA125 was identified was not stated. Using Fourier-transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry Andersch-Bjorkman et al. detected 31 peptides from a 2–3 MDa form of CA125 derived from mucus of the human cervical opening [54]. Davies et al. also reported the identification of a high molecular weight form of CA125 in human tracheal secretions [55]. Recently, we have also presented mass spectrometric data including statistical analysis and metadata of 21 peptides of a high molecular weight species of CA125 [41]. These data were used to confirm the identity of CA125 in the ascites of ovarian cancer patients as a high molecular weight protein.\nTo date, mass spectrometric data of CA125 is still quite rare and seldom reported in a way that satisfies the “Minimum Information about a Proteomics Experiment” (MIAPE) standards [56–58], even after 2007. MIAPE standards have been implemented to avoid confusion about mass spectrometric experiments as well as enhance reproducibility and reliability of MS data. It is evident that the quality of mass spectrometric data regarding CA125 varies greatly and the majority of studies do not allow for independent assessment. Furthermore, statistical analysis of the MS-generated data is seldom presented. This is a major issue, especially for CA125, as the potential for incorrect peptide matches rises with increasing length of protein sequence [59].\nAdditionally, matching experimentally found masses to theoretical masses without the usage of statistical tools is prone to produce false-positive identifications. Schober et al. demonstrated that when a mass accuracy window greater than ± 10 ppm is used, numerous peptides from different proteins with similar masses can be assigned to the incorrect protein [60]. This means that CA125 identification without supporting statistical analysis is unreliable and potentially false-positive."}