Proteomic Profiling

Suitable for complex protein sample, e.g. raw protein extract, subcellular compartment or protein complex.


About Proteomic Profiling

Proteomic profilling refer to the identification of all the proteins in the Proteome or cell. It has an origin from genomic studies in model organisms that have dominated scientific research for many decades. Continuous efforts to sequence the genome of different organisms significantly broaden our understanding of the principles of conservation and diversification of genes. Growing knowledge of the genome has directed interest to the dynamics of proteins in cell (i.e. the Proteome) as they are, afterall, the ultimate players behind every biological process.

Although there are different techniques available for protein identification (e.g. Edam Sequencing), tandem mass spectrometry arguably is the most powerful techniques. In a single experimental run, thousands of proteins could be identified. Significant insights for a biological question often obtained when these identified proteins are carefully clustered according to the nature of their biological functions. For example, one can compare the Proteomes from different cells to deduce their biological functions or discover the most abundant proteins in a specific subcellular compartment. There are many more application of proteomic profiling by MS. Interested readers could refer to here.

As the proteome of cell/tissues/organism is inherently complex, low abundance protein could not be identified because of serious suppression effect from dominant protein(e.g. cytoskeleton protein) without the use of multi-dimension chromatography step such as MudPIT.

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.

Service Package

In-solution digestion

SCX Fractionation (MudPIT)

LC-MS/MS

Raw Data Export and Conversion

Protein Identification

Free Protein Quantification

Generation of Analysis Report

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Notes

  1. Sample submitted should contains at least 200ug of proteins
  2. Sample is analyzed by Thermo LTQ-Orbitrap
  3. The default number of SCX fractions is 8. Each subsequent fraction charge US$100
  4. Proteins are identified by MASCOT® database search
  5. Free protein quantification is applicable to sample that is labeled with stable isotopes (e.g. 15N metabolic labelling or SILAC)
  6. If more than one sample are submitted, free label-free quantification service is offered.
  7. Intensity weighting, normalization, bias and isotopic overlap correction will be applied when appropriate

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