Suitable for comparing the protein level between simple protein samples. (For example, a gel band cut from 1D SDS-PAGE or highly purifed protein sample)
About Protein Quantification
Protein Quantification refer to the absolute or relative quantification of the level of proteins in one or more simple samples. The service package shown on this page is for relative quantification only. Please feel free to contact us if you require absolute protein quantification service.
Our relative quantification service consisted of one chromatography step and it is dedicated for simple protein sample only. If you need to quantify proteins in complex background, you may consider our Quantitative Proteomic Profiling service which is consisted of two chromatography steps.
Unlike electric circuit, biological system is not characterized by all-or-nothing responses. Different levels of protein or PTM (e.g. phosphorylation level) effect different phenotypes in cells. Getting protein quantification information is the clue to decipher the mechanisms behind common biological processes. By comparing the protein levels between healthy and diseased cells, for example, one can find out which proteins contribute significantly to the diseased status. Once these proteins are identified, the next step is to conduct functional studies on the protein of interest. Protein overexpression or RNA interference is one way while mutant construction is another. Confirmation of the functional role of the protein is the key for subsequent drug development. Arguably, protein quantification information is the stepping stone toward the answer of every biological wonders.
Western immunobloting is the traditional way to quantify the difference of protein levels in different samples (Figure 1). However, this method has poor resolution. While this method might be able to confidently distinguish a difference of 50%, it may not be able to do so when the difference is less than 5% that is already sufficient to kick start a signaling cascade in cell. Although mass spectrometer is a powerful device for protein identification, it is not inherently quantitative. Due to the variation of ionization efficiency and other physiochemical factors, the absolute ion intensities observed on mass spectra subject to run-to-run variability even in replicate sample. As a result, the ratio of a protein from different samples could not be accurately calculated from the ion intensities observed in spectra from separate analysis runs. On the contrary, the ratios of ion intensity on the same mass spectrum show great consistency in replicate runs (Figure 2). To obtain accurate quantification information, samples can be first labeled with different stable isotopes (e.g. iTRAQ) such that mass spectrometer could distinguish identical protein in separate samples. After the labelling, samples are mixed together and analyzed in a single analysis run.
Figure 1. Western immunobloting is used to quantify the difference of protein level in different time point for a protein.
Figure 2. Ion intensities show great inconsistency between spectra from different runs but show great consistency in terms of ratio. Both first and replicate run give a ratio of 2:1 although they have different absolute value.
iTRAQ stands for isobaric tag for relative and accurate quantitation of protein. iTRAQ is a technique, coupled with mass spectrometer, to simultaneously quantitate the difference of peptide levels between 2-8 protein samples.
iTRAQ make use of multiplexed isobaric tags to labeled the proteins/peptides from different samples independently. These tags have equal nominal mass but different isotopic contents, i.e. they are isotopologues. Derivatized peptides from different samples are pooled together and analyzed by mass spectrometer. Peptides that are identical in sequence and labelled with different isobaric tags from different samples would give the same signals (i.e. m/z values) on MS1 spectrum and each contribute a portion to the signals. This greatly enhance subsequent peptide identification because of increased precursor and hence product ion intensities. The true origin of these derivatized peptide are revealed when they are selected for fragmentation in which they produce MS/MS sequencing ions and reporter ions of the isobaric tags. Because of different isotopic composition, these reporter ions resolve to different m/z value in MS2 spectrum. A typical 4-plex labelling is resolved to 114, 115,116 and 117 m/z while 8-plex labelling is resolved to 113-119 and 121 m/z. By comparing the intensities of these peak, the peptide levels from different samples can be calculated.
Figure 1. Reaction of isobaric tag with tryptic peptide
Figure 2. Structure of isobaric tag and its isotopologues' composition
Figure 3. Structure of 4-Plex isobaric tag (114-117 m/z)
Figure 4. iTRAQ Workflow
- iTRAQ can be highly multiplexed. It can quantitate up to eight protein samples in parallel.
- iTRAQ does not increase sample complexity. This allow more proteins to be identified and quantified in single MS run.
- iTRAQ labelling is performed after proteins are extracted from cells/tissue/organism. This eliminate the need/impossibility to label protein metabolically.
- iTRAQ labelling is independent of peptide sequence when trypsin is used for protein digestion. iTRAQ react with primary amine groups on peptides. Trypsin digestion ensure at least one amine group at the terminal. Thus, no peptide is missed because it lacks certain residue e.g. cysteine as in iCAT.
iTRAQ can be applied for time-course or case-control studies. iTRAQ can be employed in phosphor-proteomic studies to reveal the level of phosphorylation in different samples. For details, release refer to our phosphoprotein identification service.
Our iTRAQ Service
N-Cell Technology offer iTRAQ labeling and analysis service. This service is based on our highly optimized iTRAQ protocol. To name a few, we use:
- Optimized 2D chromatography technique to give the best peptide identification and quantification result
- Optimized buffers for iTRAQ labelling to give the best labellng efficiency while keeping hydrolysis to minimum
- Optimized LC acetonitrile gradient to counteract the increased hydrophobicity of iTRAQ labelled peptide
- Optimized procedure to ensure the labelled sample is free of salts when injected into MS. (Noted that high salt content (>10mM) would affect subsequent chromatographic steps.)
- Optimized voltage for CID to give the best fragmentation for iTRAQ labelled peptides.
- Optimized precursor selection windows
- Optimized production of fragment ions with the best charge state
- Optimized statistic and calculation algorithm for the analysis of iTRAQ data.
Raw Data Export and Conversion
Generation of Analysis Report
- Recommended sample amount is 50-200ug for each studied condition. And make sure your sample is prepared in buffer without primary amines (e.g. not in Tris-HCl, ammonium carbonate) or detergent.
- Sample is analyzed by Thermo LTQ-Orbitrap
- Proteins are identified by MASCOT® database search
- Intensity weighting, normalization, bias and isotopic overlap correction will be applied when appropriate
- Price is based on 4-plex experiment
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