Tandem Mass Spectrometry
ITSI Biosciences offers mass spectrometry services for protein identification, protein quantitation and post translational modification (PTM) mapping. ITSI Biosciences utilizes high performance nano liquid chromatography (LC) and Tandem mass spectrometry (MS/MS) technologies to achieve unsurpassed resolution and identification accuracy. We can tailor the LC phase to analyze proteins from a simple gel plug to a complex sample like cell lysate or plasma. To increase coverage of protein identification when dealing with a complex sample like serum, plasma, whole cell lysate and tissue homogenate we utilize Multi-Dimensional Protein Identification Technology (MudPIT) to reduce sample complexity prior to LC-MS/MS.
Quantitative protein analysis is another significant area offered along with simultaneous protein identification. We can do both the label-free approach (spectral counting or ion intensity measurements) and the labeled approach using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and Tandem Mass Tags (TMT). We have successfully finished multiple projects utilizing either the label-free approach or iTRAQ and TMT based biomarker identifications and we provide results either as relative abundance (iTRAQ or TMT ratios) or absolute quantities (pmol, pg, ug or fg).
Comprehensive identification of post translational modifications (PTM) is the third most sought after service at ITSI Biosciences. We offer simple PTM analysis utilizing a multistage activation approach. For extensive PTM analysis we use either immobilized metal affinity chromatography (IMAC) or Titanium dioxide (TiO2) for phosphopeptide enrichment. For very complex samples, ITSI Biosciences starts with Strong Cation Exchange (SCX) fractionation followed by IMAC or TiO2 phosphoenrichment. For lysine rich proteins or proteins without/minimal trypsin cleavable sites, we utilize a variety of nontraditional enzymes like LysC, ArgC, AspN and GluC for good coverage and PTM results.
To ensure that proteins are sufficiently digested into peptides prior to LC-MS/MS, ITSI-Biosciences utilizes the proprietary ProDM kit to accurately calculate the percentage of proteins digested into peptides.
Our team can perform global proteomics, protein expression profiling, comparative proteomics and targeted proteomics for biomarker discovery and monitoring. Most biological and clinical samples can be analyzed directly or after electrophoresis (e.g. 2D-DIGE, SDS-PAGE) and candidate proteins can be identified by nano LC/MS/MS after in-gel or in-solution digestion.
Our Protein Identification and proteomics services include experimental design, sample preparation, tandem mass spectrometry analysis using nano LC-MS/MS, database search, identified protein annotation and report preparation with suggested next steps.
We offer the following services:
|S/N||CAT NO.||NAME||SERVICE DESCRIPTION||UNIT COST|
|1||SP1||Sample Preparation 1||Homogenization, protein quantitation using ToPA Bradford protein assay kit.||Per sample|
|2||SP2||Sample preparation 2||Includes, precipitation, buffer exchange, protein quantitation, etc. and the use of ToPI-DIGE, ToPREP and ToPA.||Per sample|
|3||SP3||Sample preparation 3||Depletion of abundant protein e.g. bovine or human serum albumin using ITSI ASK-Column kit.||Per sample|
|4||SP4||Sample preparation 4||Depletion of abundant protein e.g. bovine or human serum albumin using ITSI ASK-Solvent kit.||Per sample|
|5||SP5||Sample preparation 5||Depletion IgY Column Depletion of 14 abundant proteins from serum / plasma using IgY column.||Per sample|
|6||SP6||Sample preparation 6||Phospho-protein/ peptide enrichment using TiO2 or IMAC technology.||Per sample|
|7||SP7||Sample preparation 7||In-gel digestion. Digestion of proteins in a gel band/spot using trypsin. The service includes a) gel de-staining – coomassie or silver stain b) alkylation and reduction c) trypsin digestion d) peptide extraction from the gels for mass spectrometry analysis.||Per sample|
|8||SP8||Sample preparation 8||In-solution digestion. Digestion of proteins in solution using trypsin. The service includes; a) determining protein concentration (if possible), b) alkylation and reduction c) digestion d) peptide extraction for mass spectrometry analysis.||Per sample|
|9||SP9||Sample preparation 9||Urine protein isolation and concentration. Service includes Isolation, partial purification, and concentration of urine proteins using ITSI Urine Protein Isolation kit.||Per sample|
|10||SP10||Sample preparation 10||Zip tip cleanup of in-gel or in-solution digested samples….||Per sample|
|11||SP11||Sample preparation 11||Any other sample preparation.||Per sample|
|12||S50.FPS||Flash Proteomics Service (FPS) by tandem mass spectrometry||
The Flash Proteomics Service involves the use of validated pre-fractionation protocols and high performance tandem mass spectrometers to rapidly generate a global qualitative protein signature of a moderately-complex specimen. With FPS the run time is 30 minutes or less and most results will be ready in about 48h. Samples including fractions from HPLC, pre-fractionated samples such as protein lysates, serum, plasma and culture media can be screened. In many cases, results will be ready in 48 hours. This service is ideal for preliminary data generation, to support grant applications, biomarker research, search for contaminants in samples, verification of the integrity and purity of samples, characterization of antibodies and recombinant proteins. The standard FPS service includes:
Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of pure protein samples. Services include
Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of slightly complex samples, including proteins isolated from gel plugs and gel bands. Services include
Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of moderately complex samples. Services include
Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of very complex samples. Services include
Peptide sequencing and Post Translational Modification (PTM) Mapping of purified or partially purified proteins by Mass Spectrometry. Services include
Multi-dimensional Protein Identification Technology and peptide sequencing. Services include
|19||S80||iTRAQ based Proteomics.||
Quantitative proteomics using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ). Labeling with 2-8 tags can be used. iTRAQ Labeling is a non-gel based protein quantitation technique using 5 – 100 µg of protein. 2 to 8 samples can be compared in a single experiment. Services include
iTRAQ plus MudPIT. Service includes
|21||S90||TMT based Proteomics.||
Quantitative proteomics using Tandem Mass Tags (TMT). Labeling with 2-8 tags can be used. TMT Labeling is a non-gel based protein quantitation technique using 5 – 100 µg of protein. 2 to 8 samples can be compared in a single experiment. Services include
TMT plus MudPIT. Service includes
|23||S100||xMAP analysis||Quantitative protein analysis using Luminex xMAP technology. The level of up to 100 proteins can be measured simultaneously in 50ul of serum, plasma, tissue lysate and other body fluids.||1-100 samples|
|24||S110||Metabolomics – PMP||Primary Metabolism Profiling (PMP): This analysis targets carbohydrates and sugar phosphates, amino acids, hydroxyl acids, free fatty acids, purines, pyrimidines, aromatics, exposome-derived chemicals.||Per Sample (minimum 12 samples)|
|25||S111||Metabolomics – CLP||Complex Lipids Profiling (CLP): This analysis will target Ceramides, Sphingomyelins, Cholesteryl esters, Oxysterols, Lyso- and phospholipids, mono-, di- and triacylglycerols, galactosyl- and glucuronyllipids.||Per Sample (minimum 12 samples)|
|26||S112||Metabolomics – BAP||Biogenic Amines Profiling (BAP): This analysis will target acylcarnitines, TMAO, cholines, betaines, SAM, SAH, nucleotides and nucleosides, methylated and acetylated amines, di- and oligopeptides.||Per Sample (minimum 12 samples)|
|27||S113||Metabolomics – TMA||Targeted Metabolite Analyses (TMA): This analysis measures bile acids, steroids, eicososanoids / oxylipins and, very short chain fatty acids using liquid chromatography-QTRAP or gas chromatography-quadrupole mass spectrometry||Per Sample (minimum 12 samples)|
|28||DBS-PID||Database Search – Protein Identification||
Database search for protein identification. The service includes
|29||DBS-PTM||Database Search – Protein Identification and PTM Mapping||
Database search for protein identification and post translational modification mapping. The service includes
|30||GO||Gene Ontology (GO) Annotation||Annotation of proteins using GO classification for biological process, molecular function and cellular component.||Per search|
|31||MSI||Manual Spectrum Interpretation||Manual Interpretation of mass spectrum||Per hour (Minimum one hour)|