Large‐scale quantitative clinical proteomics by label‐free liquid chromatography and mass spectrometry

Abstract

We previously reported the development of an integrated proteome platform, namely 2-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL), for quantitative comparison of large peptide datasets generated by nano-flow liquid chromatography (LC) and mass spectrometry (MS). The key technology of 2DICAL was the precise adjustment of the retention time of LC by dynamic programming. In order to apply 2DICAL to clinical studies that require comparison of a large number of patient samples we further refined the calculation algorithm and increased the accuracy and speed of the peptide peak alignment using a greedy algorithm, which had been used for fast DNA sequence alignment. The peptide peaks of each sample with the same m/z were extracted every 1 m/z and displayed with along the horizontal axis. Here we report a precise comparison of more than 150 000 typtic peptide ion peaks derived from 70 serum samples (40 patients with uterine endometrial cancer and 30 controls). The levels of 49 MS peaks were found to differ significantly between cancer patients and controls (P < 0. T he incidence and morbidity of endometrial cancer have been rising in Western countries and Japan. In 1970, endometrial cancer constituted only 3% of all uterine cancers, but the proportion had increased to 40% by 1998.(1) The contemporary human lifestyle is characterized by excessive fat consumption, obesity, physical inactivity, high energy intake, hypertension, and a high serum glucose concentration, which are risk factors for endometrial cancer, (2)(3)(4)(5)(6) and therefore its incidence is predicted to increase further. Abnormal uterine bleeding is the most frequent initial symptom of endometrial cancer, but many other disorders including endometriosis, metroptosis, myoma uteri, and uterine sarcoma can also produce this symptom. Endometrial cancer is usually diagnosed by histological examination of endometrial tissue. However, endometrial biopsy is often associated with complications such as infection, bleeding and perforation of the uterus, and the development of an alternative safe test is therefore necessary for diagnosis of endometrial cancer.The circulating serum proteome holds great promise as a reservoir of information that will be applicable for diagnosis of various diseases. Various gel-based and MS-based quantitative analyses of plasma/serum proteins have been conducted actively to identify serum biomarkers that can be applied to blood tests.(7-10) HPLC with a flow rate of the nanoliter-per-minute order coupled with high-speed MS/MS scanning has especially attracted considerable attention because of its comprehensive protein identification capacity.(11-13) However, MS/MS is essentially not a method for quantification, and several attempts have been made to provide a quantitative dimension to nano-LC-MS/MS. Semi-quantification is possible to some extent by counting the number of peptides sequenced. (14-16) To achieve more accurate quantification, several labeling methods have been developed, (17)(1...

Functional proteomics to dissect tyrosine kinase signalling pathways in cancer

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Use of quantitative shotgun proteomics to identify fibronectin 1 as a potential plasma biomarker for clear cell carcinoma of the kidney

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“…2DICAL can accurately align different LC-MS data and compare the protein content of a theoretically unlimited number of samples without isotope labeling [7]. 2DICAL is highly advantageous methods in clinical studies that require the comparison of a statistically sufficient number of patient samples [8]. Using 2DICAL, we were able to identify diagnostic biomarkers for endometrial and pancreatic cancers [7,8] and predictive biomarkers for hematologic toxicities and therapeutic efficacy of gemcitabine treatment to patients with advanced pancreatic cancer [9].…”

Survival Prediction for Pancreatic Cancer Patients Receiving Gemcitabine Treatment

“…Two-dimensional image converted analysis of liquid chromatography and mass spectrometry (2DICAL) 1 is a new LC-MS-based proteomics platform that was developed in our laboratory (16). 2DICAL can quantify protein content accurately across a theoretically unlimited number of samples without isotope labeling and thus has considerable advantages over conventional LC-MS-based methods for clinical studies (17). The predictive biomarker protein for hematologic toxicity described above was identified using 2DICAL (8).…”