Atsuhiko Toyama

A comprehensive peptidome profiling technology for the identification of early detection biomarkers for lung adenocarcinoma.

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000–5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273–283, FIBA 5–16, and LBN 306–313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens.

Plasma low-molecular-weight proteome profiling identified neuropeptide-Y as a prostate cancer biomarker polypeptide.

In prostate cancer diagnosis, PSA test has greatly contributed to the early detection of prostate cancer; however, expanding overdiagnosis and unnecessary biopsies have emerged as serious issues. To explore plasma biomarkers complementing the specificity of PSA test, we developed a unique proteomic technology QUEST-MS (Quick Enrichment of Small Targets for Mass Spectrometry). The QUEST-MS method based on 96-well formatted sequential reversed-phase chromatography allowing efficient enrichment of <20 kDa proteins quickly and reproducibly. Plasma from 24 healthy controls, 19 benign prostate hypertrophy patients, and 73 prostate cancer patients were purified with QUEST-MS and analyzed by LC/MS/MS. Among 153 057 nonredundant peptides, 189 peptides showed prostate cancer specific detection pattern, which included a neurotransmitter polypeptide neuropeptide-Y (NPY). We further validated the screening results by targeted multiple reaction monitoring technology using independent sample set (n = 110). The ROC curve analysis revealed that logistic regression-based combination of NPY, and PSA showed 81.5% sensitivity and 82.2% specificity for prostate cancer diagnosis. Thus QUEST-MS technology allowed comprehensive and high-throughput profiling of plasma polypeptides and had potential to effectively uncover very low abundant tumor-derived small molecules, such as neurotransmitters, peptide hormones, or cytokines.

Proteomic characterization of ovarian cancers identifying annexin‐A4, phosphoserine aminotransferase, cellular retinoic acid‐binding protein 2, and serpin B5 as histology‐specific biomarkers

Numerous studies have suggested that the different histological subtypes of ovarian carcinoma (i.e. clear cell, endometrioid, mucinous, and serous) have distinct clinical histories and characteristics; however, most studies that have aimed to determine biomarker have not performed comprehensive analyses based on subtype specificity. In the present study, we performed two‐dimensional gel electrophoresis‐based differential proteomic analysis of the different histological subtypes of ovarian carcinoma using tissue specimens from 39 patients. Seventy‐seven protein spots (55 unique proteins) were found to be up‐ or downregulated in a subtype‐specific manner. The most significant difference was observed for: (i) annexin‐A4 (ANXA4) and phosphoserine aminotransferase (PSAT1), which are expressed strongly in clear cell carcinoma; (ii) cellular retinoic acid‐binding protein 2 (CRABP2), which is expressed specifically in serous carcinoma; and (iii) serpin B5 (SPB5), which is upregulated in mucinous carcinoma. Validation of these candidates by western blotting using a 34 additional test sample set resulted in an expression pattern that was consistent with the screening and revealed that differential expression was independent of cancer stage or tumor grade within each subtype. Thus, the present study reinforces the notion that ovarian cancer subtypes can be clearly delineated on a molecular basis into four histopathological groups, and we propose that ANXA4, PSAT1, CRABP2, and SPB5 are candidate subtype‐specific biomarkers that can help define the basis of tumor histology at a molecular level. (Cancer Sci 2012; 103: 747–755)

Deglycosylation and label-free quantitative LC-MALDI MS applied to efficient serum biomarker discovery of lung cancer

BackgroundSerum is an ideal source of biomarker discovery and proteomic profiling studies are continuously pursued on serum samples. However, serum is featured by high level of protein glycosylations that often cause ionization suppression and confound accurate quantification analysis by mass spectrometry. Here we investigated the effect of N-glycan and sialic acid removal from serum proteins on the performance of label-free quantification results.ResultsSerum tryptic digests with or without deglycosylation treatment were analyzed by LC-MALDI MS and quantitatively compared on the Expressionist Refiner MS module. As a result, 345 out of 2,984 peaks (11.6%) showed the specific detection or the significantly improved intensities in deglycosylated serum samples (P < 0.01). We then applied this deglycosylation-based sample preparation to the identification of lung cancer biomarkers. In comparison between 10 healthy controls and 20 lung cancer patients, 40 peptides were identified to be differentially presented (P < 0.01). Their quantitative accuracies were further verified by multiple reaction monitoring. The result showed that deglycosylation was needed for the identification of some unique candidates, including previously unreported O-linked glycopeptide of complement component C9.ConclusionsWe demonstrated here that sample deglycosylation improves the quantitative performance of shotgun proteomics, which can be effectively applied to any samples with high glycoprotein contents.

Potential Implications for Monitoring Serum Bile Acid Profiles in Circulation with Serum Proteome for Carbon Tetrachloride-Induced Liver Injury/Regeneration Model in Mice

Bile acids have recently emerged as versatile signaling molecules, and their signaling pathway is a promising target for the treatment of metabolic diseases. Here, we developed a highly sensitive and high-throughput quantification method for six taurine- and glycine-conjugated bile acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after solid-phase extraction (SPE-MALDI-TOF MS). In a carbon tetrachloride (CCl4)-induced liver injury/regeneration model in mice, serum bile acid profiles were monitored, and the same samples were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and protein spots that significantly changed in quantity in a serial time points were identified by MALDI-TOF MS. Serum taurocholic acid (TCA) concentration was significantly elevated earlier than the increase of serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT) activity, a potentially sensitive marker for minimal hepatic damage. Furthermore, TCA peaked at 20 h after treatment when massive serum proteins appeared in circulation. It should be noted that direct MALDI-imaging mass spectrometry (IMS) has succeeded in showing a hepatic lobular distribution change of TCA, predominantly seen in zone 1 area whereas necrotic changes were dominant in zone 3 area. The in-depth analysis of bile acid profiles in circulation with hepatic lobular distribution is a strong basis to understand the serum proteome in CCl4-induced liver injury model.

Direct antigen detection from immunoprecipitated beads using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry; a new method for immunobeads‐mass spectrometry (iMS)

One-step detection of biological molecules is one of the principal techniques for clinical diagnosis, and the potential of mass spectrometry for biomarker detection has been a promising new approach in the field of medical sciences. We demonstrate here a new and high-sensitivity method that we termed immunobeads-mass spectrometry (iMS), which combines conventional immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The key feature of iMS is the MS-compatible condition of immunoprecipitation using detergents with a monosaccaride-C8 alkyl chain or a disaccharide-C10 alkyl chain, and the minimized number of steps required for high-sensitivity detection of target peptides in serum or biological fluid. This was achieved by optimizing the wash buffer and subjecting the immunobeads directly to MALDI-TOF MS analysis. Using this method, we showed that 1 fmol of amyloid beta peptide spiked in serum was readily detectable, demonstrating the powerful tool of iMS as a biomarker detection method.

Abstract 3214: Definition of biosimilars: Energy Resolved Oxonium Ion Monitoring (Erexim) technology grasps detailed N-glycan microheterogeneity on therapeutic antibodies.

The patent expiry of innovative antibody drugs has provoked a great deal of concern over generic drugs. Reduction of medical costs by generics is undoubtedly beneficial to all countries, however, major issues about bioequivalence and immunogenicity remain incompletely addressed. The first draft guidance on the evaluation of biosimilarity was published by FDA in Feb 2012, in which emphasis was placed on the importance of evaluating minor structural differences that can significantly affect the potency and safety of biopharmaceuticals, with specific reference to glycosylation patterns on antibody drugs. We propose here a new method for validating the microheterogeneity of N-glycans on therapeutic antibodies. The continuous collision energy scanning of oligosaccharide-derived fragment ions (oxonium ions) illustrated both comprehensive structural information of glycans on glycopeptides and their quantitative abundance ratio simultaneously in a single analysis of a triple quadrupole mass spectrometer. We showed that oxonium ion m/z 138 could be a specific reporter ion for sensitive quantification of glycopeptides, regardless of glycan structures. Indeed the quantitation limit of glycopeptide was 30 attomole and, importantly, even very rare structural isomers were quantifiable, which existed only 0.1% of all glycoforms. We named this technology Energy Resolved Oxonium Ion Monitoring (Erexim) and utilized for the evaluation of lot-to-lot glycoform variations of monoclonal therapeutic antibodies trastuzumab and bevacizumab. The quantitative profiles of 4 lots (n = 4 each) revealed that N-glycans on both drugs showed gradually shortened tendency with time. Surprisingly some abundant glycoforms on both antibody drugs exhibited 10% changes between two continuous lots. We further applied Erexim technology to in-depth analysis of cetuximab glycosylation pattern, previously known to induce severe anaphylaxis. Our method successfully provided a comprehensive structural view of Fab- and Fc-glycans simultaneously in 30 minutes analysis, which uncovered lots of critical non-human type antigens only on the Fab region of cetuximab, such as Gal(α1-3)Gal or Neu5Gc structures. Hence in the process of future biosimilar approval, the Erexim technology we demonstrated herein has potential to define “how similar it is”. Citation Format: Koji Ueda, Atsuhiko Toyama, Taka-Aki Sato, Yusuke Nakamura, Hidewaki Nakagawa. Definition of biosimilars: Energy Resolved Oxonium Ion Monitoring (Erexim) technology grasps detailed N-glycan microheterogeneity on therapeutic antibodies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3214. doi:10.1158/1538-7445.AM2013-3214 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 4569: Serum proteome analysis by label-free quantification system and LC-MALDI mass spectrometry for the discovery of novel biomarkers for lung cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is the leading cause of cancer death in Japan and throughout the world, and highly specific and sensitive serum biomarkers applicable for screening are urgently required. Here we report our label-free quantification study conducted on the serum samples from 10 healthy volunteers, 10 patients at early stages and 10 patients at advanced stages of lung adenocarcinoma, aiming to identify serum proteins that can be used as lung cancer biomarkers. The workflow was divided into four sections: 1) serum sample preparations involving immunodepletion of 14 high-abundance proteins, enzymatic deglycosylation in H218O, trypsin digestion and nano-reversed phase liquid chromatography; 2) acquisition of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) data from 30 cases; 3) label-free quantification and statistical analysis using Genedata Expressionist® platform; and 4) candidate biomarker protein identification by retrospective MS/MS acquisition. Deglycosylation was the critical step in the sample preparation phase as it could reduce spectral complexity caused by the presence of glycopeptides with heterogeneous glycan structures. Here we utilized H218O to incorporate 18O into aspartic acid residues that occurred during the deglycosylation reaction, and thus the deglycosylation sites resulted in the specific +3 Da mass shift allowing unambiguous determination of deglycosylated peptides and their glycosylation sites. We have shown here that 7.5 % of the identified peptides were N-glycosylated. This high level of glycopeptide occurrence, attributable to the fact that over 90 % of proteins in serum are glycoproteins, suggests that deglycosylation process would significantly simplify the peptide composition, which is critical for in-depth mass spectrometric quantification analyses of serum samples. As the result of label-free quantification analysis, approximately 15,000 peptide species (as peak clusters) were detected from 30 serum samples. 25 peptides were screened by one-way analysis of variance to be differentially represented among the three sample groups (normal, early and advanced lung cancer cases, p < 0.01). 16 peptides were specifically detected in normal serum and 9 showed increased levels in advanced stage cases, of which 2 were also detected at higher levels in early-stage cases than normal sera (p < 0.05). These peptides were subjected to MS/MS acquisition, referring back to the original data to select maximally detectable spot on the archived MALDI target plate for increased chance of identification. In conclusion, the combination of H218O deglycosylation, MALDI MS and label-free quantification can work effectively for precise quantification of glycoprotein-rich clinical specimens, allowing for the screening of serum biomarkers for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4569.