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(Relative quantification mainly hereafter)
Our main tools for quantitative proteomic analysis or differential protein expression analysis are Isobaric labeling based technologies, such as iTRAQ, ICAT, SILAC and TMT.
iTRAQ Protein Quantification Service: iTRAQ is a non-gel based multiplexed protein quantitation technique that provides relative and absolute measurements of in theory all peptides from different samples/treatments.
iTRAQ is ideally suited for comparing normal, diseased, and drug-treated samples, time course studies, biological replicates and relative quantitation. Quantitation of differences in protein amount between different samples can be done by iTRAQ isotope tagging of peptides and 2D-LC separations (up to 8 separate samples with 600-1200 proteins identified and quantitated in a single experiment).Applied Biosystems iTRAQ isobaric affinity labels allow for multiplexing up to 4 or 8 samples (4-Plex or 8-Plex) in a single experiment.
The structure of the iTRAQ® reagent is shown in Figure 1. The labeling reagent consists of a quantification group (N-methylpiperazine), a balance group (carbonyl), and a hydroxyl succinimide ester group that reacts with the N-terminal amino groups of peptides and the amino groups of lysine. Furthermore, the isobaric tag consists of a balance group and a reporter group. There are eight kinds of iTRAQ® reagents, each having stable isotopes that are uniquely distributed between the balance and reporter groups. Identical peptides obtained from different samples can be labeled with different iTRAQ® reagents to be used as iTRAQ®-modified peptides having the same mass (Figure 2). When iTRAQ®-modified peptides are analyzed by MS/MS, the ratios of peptide quantities among different samples are expressed as signal intensity ratios of the reporter groups (m/z: 113, 114, 115, 116, 117, 118, 119, and 121).
After 1) denaturation, reduction and alkylation, 2) enzyme digestion, and 3) iTRAQ® modification, multiple peptides are labeled, including peptides with post translational modifications. Each sample will be labeled individually, and they will be pooled and fractionated into up to 25 or 30 fractions by SCX. Nano LC-MS/MS using latest HCD technology in OrbiTrap or Q-TOF will be performed on each fraction (Figure 3). Identical peptides derived from different samples have the same mass. These peptides share similar chromatographic properties, allowing both peptide identification and quantification to be derived from the same MS/MS spectrum. iTRAQ chemistry offers more reliable quantification than other shotgun approaches that require highly reproducible chromatography runs. In MS/MS analysis, the signal intensity ratios of the reporter groups indicate the ratios of the peptide quantities and can be used to determine the relative quantities of the peptides.
The MS/MS spectra of the individual peptides show signals reflecting amino acid sequences and also show reporter ions reflecting the protein contents of the samples. A database search is then performed using fragmentation data to identify the labeled peptides and hence the corresponding proteins whilst the iTRAQ mass reporter ion is used to relatively quantify the peptides. Quantitation of protein from multiple samples can be achieved in the same run.
Figure 1
Figure 2
Figure 3
We are developing approaches in which iTRAQ is combined with specific enrichment strategies, including enrichment for phospho-peptides, to quantify changes in the modification level of proteins. Furthermore, we develop approaches and techniques to improve the sensitivity of iTRAQ analysis. This includes specific sample handling steps to reduce the sample amount required and the implementation of exclusion lists in order to mine further down into the proteome. These exclusion lists are created from the first analytical round of data, which will be applied as a filter for re-analysis of the remaining sample. Previous experiments with the use of exclusion lists have resulted in the identification of approximately 50% more proteins than in the initial analyses.
Advantages of our iTRAQ technology include:
- Ideal for comparative studies: Up to 4 or 8 labels can be used for multiplexing experiments
- The ability to both identify and quantify the detectable elements of a proteome in a relatively short period of time (when compared to 2D gel electrophoresis which is tedious);
- Non-targeted approach for discovery studies
- Large dynamic range, high and low abundant proteins are identified
- Thousands of protein peptides can be identified and quantified in one analysis
- Post translational modifications can be detected
- Relative quantitation
- SOP-driven work flow
- Established Quality Control procedures
Our iTRAQ relative quantification analytical package includes: Extraction of Protein + Reductive Alkylation & Post-processing + Digestion + MALDI-MS + iTRAQ kit & Labeling + Mix Together Samples of Same Group+ SCX HPLC + Desalting + LC-MS/MS+ Searching + Identification and Quantification of The Proteins +Report with Intensive Data Interpretation & Data. We have extensive experiences in both technologies and will work with you from any steps in the workflow. Please contact us for a free project consultation.
For this kind of analysis, the researcher should submit from 100ug-200ug of proteins for each studied condition. iTRAQ labeling requires proteins to be present in a solvent containing neither primary amines (such as Tris, ammonium bicarbonate, etc.) nor detergents.
However, because common protein extraction methods often fail to meet these criteria, the received samples are generally submitted to protein precipitation using acetone prior to resolubilization in a compatible buffer and further analysis.
Any proteomic analysis is a challenge due to the huge dynamic range of protein concentrations in a cellular extract. Unlike nucleic acid molecules which can be amplified, proteins are detected by the mass spectrometer according to their initial concentration in the extract. In this context, the most abundant proteins will monopolize the mass spectrometer detector at the expense of proteins of lower abundance.
It is therefore important to keep in mind that the more a cellular extract is fractionated, the better are the chances to identify low abundance regulatory proteins. Analysis of crude extract will mostly yield identifications of proteins from the cytoskeleton or implicated in the intermediary metabolism.
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