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A. Proteome Profiling

Proteome profiling can identify the maximum amount of protein species from a complex sample extract, such as tissue, plasma, and body fluid. Based on mass spectrometry (MS) technology, it can supply a satisfactory sample size to serve as a foundation for future quantitative studies or modification studies of target proteins.

Technical Advantages
  • High throughput:

    Tens of thousands of proteins can be identified with a high degree of automation, compared to 2D-PAGE;

  • High accuracy:

    Based on mass spectrometry with a mass accuracy of better than 1 ppm, the credibility of proteins identified can be improved in complex samples by lowering the false positive rate;

  • High resolution:

    The resolving power is a minimum of 100,000 FWHM, producing more accurate results.

Study Workflow

B. Protein Identification

Mass spectrum based protein identification aims to identify the maximum amount of protein from gel samples or those with a low to moderate protein complexity. By combining the gel based separation system and mass spectrometry (MS) technology, Gel band identification is broadly used to identify proteins in samples with low protein complexity, such as IP samples, proteins with a specific molecular weight, etc. Gel spot identification is used to identify the proteins in a 2DE gel spot containing a single or several proteins.

Standard workflow for MS analysis

C.Post-Translational Modification Proteomics——phosphoproteome identification

Protein post-translational modification (PTM) increases the functional diversity of the proteome either by the covalent addition of chemical moieties or functional groups, or by the proteolytic cleavage of regulatory subunits or the degradation of protein complexes. Many large-scale post-translational modification studies have recently been performed on various organisms, and phosphorylation, acetylation, methylation, and glycosylation are among the most intensively studied PTM proteomes.

Protein phosphorylation is an ubiquitous post-translational modification, essential to many physiological and biological processes and cellular events. Phosphoproteomic analysis provides identification of phosphorylated proteins and peptides, providing key data for understanding their biological functions. This proteomic method has greatly enhanced our understanding of cellular phosphoproteins and their dynamic regulatory mechanisms in diverse organisms. Profiling of phosphoproteins in relation to different cellular events has enabled us to establish phosphor-relay networks in different cellular signaling.

Phosphoproteomics is a protein analytical process and high-throughput technology, by which a temporal and spatial dynamic phosphoproteome of organisms can be profiled through sample preparation, phosphopeptide enrichment, LC-MS/MS analysis, and bioinformatics analysis.

Advantage
  • High accuracy:

    Capable of localizing a phosphorylation site to a specific amino acid;

  • Large-scale:

    Hundreds of thousands of phosphosites can be measured in a single experiment;

  • Multiple fragmentation methods:

    Provides CID, HCD, or ETD fragmentation methods.

Study Workflow

D. Quantitative proteomics

Quantitative proteomics is a powerful method of discovering proteins that are expressed differently across time, disease state, or other conditions. Quantitative proteomics can be applied in many research areas, such as developmental biology, disease mechanisms, drug target discovery, biomarker screening, disease-resistant breeding, and in the study of pathogens, functional microbes, etc.

Table 1 Comparison of mainly quantitative proteomics techniques
Technology 2-DE DIGE 15N labeling SILAC ICAT iTRAQ Label free
Samples type Protein with high abundance Cultural cells Cultural cells All samples All samples All samples
Throughput 1-2samples 2samples 2-3samples 2samples 2-8samples unlimited
Proteins identified + ++ ++ ++ ++ +++
Dynamic range + ++ ++ ++ ++ +++
Quantitative accuracy + ++ ++++ ++ +++ ++
Cost + +++ +++ ++ ++ +++
Advantage of LC-MS based quantitative proteomics
  • Large-scale:

    Compared to traditional two-dimensional electrophoresis, LC-MS/MS can identify hundreds and thousands of proteins simultaneously and detected dozens or hundreds of differentially expressed proteins;

  • High throughput:

    Eight or more samples can be compared at a time;

  • High degree of automation:

    Liquid chromatography and mass spectrometry is used in conjunction with automated operations enabling rapid analysis;

  • High resolution:

    The resolving power is a minimum of 100,000 FWHM, producing more accurate results;

  • Comprehensive:

    All proteins expressed in each sample can be compared, which gives a comprehensive view of pathological mechanisms, physiological phenomena, or responses to biological/abiotic stress.


E. Targeted Proteomics Analysis

Targeted proteomics, based on multiple reaction monitoring, is able to selectively detect and quantify special function-related proteins in a large sample with the aim of inferring their molecular function. Multiple reaction monitoring (MRM) is a highly sensitive and selective method for quantifying target proteins, and currently draws the attention of many researchers worldwide, as it provides a robust and accurate measurement of protein profiles.

The MRM technique solves the problem of time consuming immunological methods and has greatly reduced the verification and validation time in the research into protein biomarkers. Compared to the antibody technique, which measures a protein using an antibody, the MRM technique enables researchers to study tens and hundreds of targeted proteins at one time. In light of these advantages, MRM technique has been widely used to verify disease related biomarkers, the detection of low-abundance proteins in highly complex mixtures, and the study of protein post-translational and chemical modification.

Application of MRM
1) Verification for protein biomarker

The discovery and verification of biomarkers by proteomic methods is currently drawing much research attention. The research process of “discovery–verification clinical validation” has been widely accepted. During the discovery period, the quantitative proteomic technique is used to find the differential expression proteins from disease and control samples. The MRM technique is then used to verify and quantify the expression of the targeted proteins in various samples, and finally the verified potential biomarkers are validated in a large number of clinical samples. This research pattern for the discovery of disease biomarkers is robust and cost-effective.

2) Post-translational modification of protein

Post-translational protein modification is key to gene expression and regulation. The elucidation of post-translational protein modification will help us understand the molecular basis of life and the disease process. Normally, digested peptides need to be enriched to enhance sensitivity and improve results, but achieving a satisfactory result is still difficult, and much important information is lost due to low sensitivity. The high sensitivity of targeted proteomics is a unique advantage in studying the post-translational modification of protein.

3) Low abundant protein

Wide dynamic range and the high complexity of proteins in blood, serum, or other body fluids severely affect the accuracy of protein quantification. Though investigators try many methods of to deal with these problems, such as eliminating the high abundant protein and prolonging the analysis time of HPLC, the sensitivity and accuracy of mass spectrometry still cannot meet the requirements for the detection of low abundant protein. With the improvement of mass spectrometry, Multiple Reaction Monitor- based targeted proteomic could help to achieve this goal due to its excellent range. It broadens the detection range and increases the detection sensitivity for low abundant protein and also balances the signal difference of transitions between high and low abundant protein.

Advantages
  • High selectivity:

    Capable of detection targeted protein, avoiding the effect of low abundance on detection;

  • High throughput:

    Dozens of target proteins can be measured at one time;

  • High sensitivity:

    Can selectively analyze protein, eliminate the interference of background noise, and accurately quantify the protein.

The basic workflow of MRM