Quantitative Proteomic Profiling

Suitable for comparing the protein level between complex protein samples. (e.g. raw protein extract)


About Quantitative Proteomic Profiling

Quantitative Proteomic Profiling refers to the identification and quantification of all the proteins in the proteome. As the proteome of cell/tissues/organism is inherently complex, low abundance protein can only be identified when multi-dimension chromatography technique such as MudPIT is used to reduce sample complexity and the suppression effect from dominant proteins(e.g. cytoskeleton protein). Our Quantitative Proteomic Profiling service consisted of two chromatography steps. Complex samples are fractionated by strong cation exchange into 8 fractions each of which is further fractionated by reverse phase liquid chromatography before injecting into MS for protein identification and quantification.


Protein Digestion

Protein Digestion

Proteins are digested with proteolytic enzyme.

Peptide Separation

Peptide Separation

Digested peptides are fractionated by RPLC

Tandem MS Analysis

Tandem MS Analysis

Each fraction is on-line injected into MS

Data Analysis

Data Analysis

Peptide sequences are obtained from MS2 spectra


With the use of high resolution LC/MS/MS, the exact sequence of a protein can be revealed on MS2 spectrum, Figure 1 and 4. In typical experimental workflow, the protein of interest is first digested and downsized with proteolytic enzyme into small peptides which are more readily resolved by mass spectrometer. These peptides are then separated based on their hydrophobicity in a reverse phase liquid chromatography column. Each chromatographic fraction is analyzed by MS. In MS1, the peptides in a fraction are separated based on their mass-to-charge ratio to obtain MS1 spectrum. Given sufficient resolution of mass spectrometer and free of other interference, an elegance isotopic envelope of a peptide could be observed, Figure 2. If the peptide has low molecular mass, the most abundant peak would be the monoisotopic peak. In data-dependent acquisition mode, the most abundant peak/peaks would be selected for further fragmentation. If collision induced fragmentation method is chosen, the monoisotopic peptide would be fragmented into a series of y- or b- ions, Figure 3. These ions are then separated in MS2 based on their masses. By calculating the mass difference between adjacent y- or b- ions respectively, the peptide sequence could be obtained as amino acids have different exact masses (except isoleucine and leucine), Figure 4



Figure 1. Peptide sequence is revealed on MS2 spectrum.

Figure 2. Typical isotopic envelope of peptide.

Figure 3. CID produces a series of y- and b-ions.

Figure 4. Peptide sequence can be obtained by comparing the mass difference between adjacent y- or b-ions respectively with monoisotopic masses of amino acid

MudPIT stands for Multi-Dimension Protein Identification Technology. Compared to single dimension chromatography, multi-dimension LC-MS/MS chromatography can greatly increase resolution and loading capacity for identifying large numbers of proteins with board dynamic range of protein abundance. Although many multi-dimensional separation techniques have been published, the combination of strong-cation exchange (SCX) with reversed-phase (RP) chromatography has shown the most promising result. In contrast to traditional gel-based separation method, chromatography based SCX-RP method guarantee high throughput and ease of automation.

In a typical analysis workflow, protein sample is first digested enzymatically into smaller peptides and then fractionated by strong cation exchange chromatography column. Peptides with higher positive charges bind more strongly to the column. Peptides are eluted from the column using a gradient of salt. Each collected fraction is subjected to second round of fractionation by reverse-phase chromatography. Hydrophobic peptides bind more strongly to the column. Peptides eluted from the column are ionized and injected into MS/MS for analysis. Acquired MS2 spectra are compared to protein sequences in either protein or translated nucleic acid database. Peptides that are confidently matched with sequences in the databases are used to reconstitute a list proteins in the protein sample. This methodology normally can identified hundreds or thousands of proteins depending on the sample complexity.

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.


Service Package


In-solution/gel digestion

iTRAQ Labelling

SCX Fractionation (8 fractions)

LC/MS/MS Analysis

Raw Data Export and Conversion

Protein Identification

Protein Quantification

Generation of Analysis Report


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  1. Sample submitted should contains at least 200ug of proteins
  2. Sample is analyzed by Thermo LTQ-Orbitrap
  3. Proteins are identified by MASCOT® database search

Quantitative Proteomic Profiling