Mass Spectrometry play a major role in protein identification, protein quantitation and post translational modification (PTM) mapping, we 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. 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, usually 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 following services
Protein Identification and profiling
Protein Intact Mass Analysis
TMT-based Quantitative Proteomics
TMT-MudPIT Quantitative Proteomics
xMAP for Quantitative Protein Analysis
A peptide centric (bottom up) approach using nanoLC-MS/MS and multiple modes of fragmentation (CID, HCD, ECD) to identify the amino acid sequences of peptides.
Samples from solution or polyacrylamide gels are digested using trypsin (alternative enzymes available); peptides are purified, concentrated, and loaded onto a nanoLC C18 analytical column. Read more
Protein Differential/Expression Analysis
Relative Qualitative and Quantitative approaches indicating protein levels within the same sample as well as differential levels among samples. A variety of approaches exist within this category. Modern proteomics dictates that protein identification is only part of the puzzle, and increasingly it is necessary to understand the expression profiles of protein(s) within the overall context of the cell. Multiple solutions exist, some more accurate, robust and applicable than others. Our lab has experience with several methodologies including. Read more