iTRAQ (4 plex or 8 plex) and TMT (2 plex through 10 plex) are chemical labeling methods that enable high through-put and internally controlled quantifications in proteomics studies. This is a very popular service in Complete Omics which enables researchers to directly compare the difference of proteomes in their treated vs untreated, diseased vs normal, over-expressed vs control, gene silenced vs control, knockout vs wild type, mutant vs wild type groups, etc.
Every sample group in the study is normalized to start from the equal amount of total protein/peptide. Protein digest of each sample is chemically labeled with a sample specific tag. Tagged peptides from different samples are pooled together with equal total individual amount and the complete catalogue of proteins in the pooled sample is resolved by Mass Spectrometry analysis and each protein identified in the study will contain 4 or 8 (for iTRAQ) and 2 through 10 (for TMT) intensity values representing this protein’s abundance in different samples. The result shows not only the identification of peptides/proteins, but also the absolute/relative abundance of the peptides/proteins in each sample.
Your samples (several test groups and control group) will be digested with Trypsin and peptides are chemically labeled by either iTRAQ or TMT labeling method, and the labeled peptides are pooled and ion-exchanged through Strong Cation Exchange (SCX) HPLC cleaning. To improve the sensitivity, we suggest the basic Reverse Phase LC (bRPLC) to further fractionate your cleaned sample into 96 fractions and then pooled to 24 fractions to maximize the coverage and depth of your analysis. 24 Mass Spectrometry Analysis runs will be performed by the world’s most advanced Orbitrap LC-MS/MS system.
Data analysis, including data validation, visualization and quantification, are performed with commercial softwares and Complete Omics’ unique R packages and scripts. Report will be sent to you in Excel format as well as a summary in PDF format. We will also provide you any details you need for your papers’ MATERIALS AND METHODS section. We will make sure you understand your result and help you with your paper writing with free follow-up services.
Sample types we accept:
1, 2D gel spots, SDS-PAGE bands
2, Cell Lysates and Tissue Lysates
3, Biofluids, such as plasma*, serum*, saliva, tear, etc.
*We provide High Abundance Protein Depletion Service to significantly (100-500 folds) increase the depth of your biofluid proteomics analysis by removing top abundant proteins from your samples. Read more for details.
4, FFPE slide/ FFPE extract
5, Customized sample types (please contact us to discuss)
Detecting and Absolutely Quantifying Patient-specific Neoantigens from Limited Input of Biopsy Sample — An Integrated Pipeline for Neoantigen-targeted Cancer Treatments
Neoantigens derived from HERVs represent a new group of cancer therapeutic targets. In a collaborating project, Complete Omics identified and quantified HERVs derived neoantigens and provided solid evidence for developing…
Some of our impacts
2022--Direct Identification and Quantification of Neoantigens from Minute Amount of Clinical Biopsy Sample --- published on CancersIn recent years, neoantigens are becoming popular cancer therapeutic targets under intensive studies by almost all major oncology pharmaceuticals. However, who are the target patients? Does the patient with a "correct" mutation and a "correct" HLA allele indeed present the "correct" neoantigen? Is this individual's neoantigen copy number high enough for immunotherapy? AI predictions based on NGS genomic information have been proven incapable of answering these questions. Immunopeptidome through mass spectrometry is dominated by disease-irrelevant peptide sequences. There is no existing way to identify and quantify neoantigens from a minute amount of clinical biopsy sample, such as 50 mg tissue or less. Valid-NEO is developed to fit this demanding clinical need through combining our proprietary multi-omics platforms including NGS-based ultra-rare mutation calling technique, DEEPER-SeqS, and our unique clinical proteomics platform, Complete360®, with additional hardware innovations (Ref. 1).Valid-NEO: Multi-omics Pipeline for Neoantigen Assays
2022--Identify and Quantify Neoantigens derived from Human endogenous retroviruses (HERVs) --- published on Science AdvancesHuman endogenous retroviruses (HERVs) represent 8% of the human genome. Working with, ErVaccine Technologies, our scientists identified neoantigens encoded by HERVs across a broad spectrum of cancers, and this finding may help enable next-generation therapeutic vaccines and cellular immunotherapies targeting these so-called “unconventional” tumor antigens (Ref 1). These antigens are shared by different tumor types. They would prove useful as personalized cancer therapeutic targets for a large number of patients. Along the side when we are keep on improving and developing our own disease detection and companion diagnostic pipelines, we are excited to be working with a large number of collaborators to implement our multi-omics platforms in their different clinical and basic research projects.Identify and Quantify Neoantigens derived from Human endogenous retroviruses (HERVs) — A new class of cancer therapeutic targets
2021--Therapeutic Neoantigens Encoded by Oncogene K-Ras --- published on Science ImmunologyK-Ras is one of the most highly mutated oncogenes in cancers. The neoantigens encoded by K-Ras can be presented by many different types of cancer cells. Here we utilized our multi-omics neoantigen validation pipeline to detect and quantify K-Ras neoantigens from a variety of cancer samples (Ref 1.). We found that the K-Ras as well as several other oncogenes can be presented on cancer cell surface, but at extremely low copy numbers. We adopted a variety of internal control system to measure their abundance down to <1 copy per cell level. To come up with an actionable strategy, the therapeutic team at JHU developed bispecific antibodies that can target the neoantigens we identified, and deliver dramatic therapeutic effects to mice. These findings are significant in the way that it opens a gate to developing pan-cancer immunotherapeutic agents that can treat a large number of patients sharing cancer hotspot mutations. Identifying such neoantigens is the first step in this campaign and is being accomplished by Complete Omics Inc.K-Ras Neoantigen Identified for Personalized Cancer Therapeutics
2021--Direct Quantification of Neoantigens Like Never Before --- published on ScienceGenetic changes in human genome are the driving force for all cancers. Different patients have different sets of mutation profiles even for the patients who all have the same disease. For decades, doctors, cancer researchers, and pharmaceutical companies have been working tirelessly trying to find a way to treat each person's unique disease in a highly personalized way that will reach the maximum treatment efficacy with the lowest side effects. Complete Omics, working with leaders in cancer therapeutics, has developed pipelines based on our multi-omics techniques through which we clearly observe and quantify personalized therapeutic targets encoded by the most frequently mutated tumor suppressor gene TP53. We validated and quantified the TP53 neoantigens on the surfaces of cancers and provided information to healthcare providers to support their decision on if or not to adopt a highly personalized cancer treatment targeting these neoantigens and when to use it (Ref 1). These findings provided the 1st-hand evidence for cancer therapeutics without the uncertainty that comes with predictions.Prediction-FREE Neoantigen Validation Enables TP53-targeted Personalized Cancer Therapeutics
2019--First Pipeline for Direct Detection and Quantification of Neoantigens --- published on Cancer Immunology ResearchCancer-linked genetic mutations can code for mutant proteins, which can then be processed by proteasomes into peptides that are presented by human leukocyte antigen (HLA) molecules, triggering the body's immune response. The idea that such mutant peptides can trigger an immune response is fundamental to immunotherapies like checkpoint inhibitors as well as cancer vaccines that present the body with these peptides to generate an immune reaction. However, while the rise of next-generation sequencing has allowed researchers to identify a large number of cancer-linked mutations, actually detecting these mutation-associated neo-antigens, or MANAs, at the peptide level remains difficult. The fact that a mutant is present at the genetic level does not mean it will be produced at the protein level, and, even if it is, that is no guarantee that it will be processed and presented by HLA molecules. This has proved a challenge for, for instance, personalized cancer vaccine development. Our technology (Ref. 1) provide the 1st pipeline for detecting personalized cancer therapeutic targets. Our method has been reported by public media and has a significant impact in cancer research (Ref. 2).Direct Detection and Quantification of Neoantigens