Protein modification analysis service_Wuhan biological Optical Valley Science and technology

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Protein modification analysis service

Product Description

Case Analysis

Result display

Sample delivery

Q&A

The research work of proteomics not only focuses on the changes of protein expression levels in different cell growth periods or under disease conditions, but also many vital life processes are not only controlled by the relative abundance of proteins, but also regulated by spatiotemporal and spatiotemporal distribution of reversible post-translational modifications. Therefore, revealing the rules of post-translational modifications is an important prerequisite for analyzing the complex and diverse biological functions of proteins. The molecular weight of protein will change when it is modified after translation. The molecular weight of protein or polypeptide can be accurately determined by mass spectrometry. At the same time, the content of post-translational modified protein in the sample is low and the dynamic range is wide, so it is necessary to enrich the modified protein or peptide segment before mass spectrometry detection.
After the protein is enzymatically hydrolyzed into peptide segments, the peptide segments are labeled with TMT, and then the corresponding modified peptide segments are enriched selectively with protein-modified pan-antibodies or biomaterials, and finally the quantitative analysis of samples is completed with high-resolution mass spectrometry.

Phosphorylated proteomics research process

Modification types: phosphorylation, acetylation, ubiquitination, methylation, etc.
Instruments used: THERMO: Orbitrap Eclipse, Orbitrap Explorer 480, Q Active plus high resolution mass spectrometry.
Technical advantages: the relative abundance of various modifications in 2-10 samples can be compared in parallel; Using high-quality modified pan-antibodies and highly selective bioconcentration materials, the identification flux of modification is high; It can be used for various types of samples.
Application fields: disease mechanism research, personalized medical treatment, agricultural variety improvement.

Sample requirements：

Sample Type	Sample requirements (per group of samples)
Protein extract	Concentration >1μg/μL, total protein >300μg
Cell sample	Cell volume>107
Tissue samples	Animal, microbial tissue wet weight >10mg; plant fresh tissue >100mg
Body fluid sample	Blood volume>500μL

　　Lead time：

Progress	Time period
Sample collection/Quality control	1-2 working days
Sample processing	4-5 working days
Mass spectrometry test	2-3 working days
Data analysis	2-3 working days
Total	No more than 20 working days

Note: For more than 4 samples, 5 working days will be added for each additional sample. If there is an urgent need, please contact us for negotiation.

Project advantages

a. Advanced instruments: We have THERMO Orbitrap Exploris 480, THERMO Q Exactive plus, THERMO Orbitrap Eclipse and other high-resolution liquid mass spectrometry systems, which have the advantages of high resolution, high scanning speed and high quality accuracy.

THERMO Orbitrap Exploris 480

THERMO QE plus

THERMO Orbitrap Eclipse

b. Professional technical team: We provide full range of proteomics-related services such as protein qualitative analysis, protein quantitative analysis and protein modification analysis, with stable experimental protocols and strict quality control standards to ensure that your experiments achieve optimal results.

c. Customized services: With professional bioinformatics team and large computers, we can provide customized bioinformatics analysis services for our partners.

d. Complete after-sales service: From report interpretation to article publication, Wuhan BioBank could provide efficient and high-quality services throughout the whole process.

Case 1 PhoP degradation mechanism in Salmonella depends on tyrosine phosphorylation

Project Introduction

A Gram-negative bacterium was used for the study material. There is a very well-known systems in bacteria which could sense external signals and make responses, called binary regulatory systems. The basis of binary regulatory system signaling is protein phosphorylation. One of the most critical is the PhoP/PhoQ system, in which the receptor protein PhoQ on the cell membrane responds to external signals by self-phosphorylation and then activates the regulatory protein PhoP by transferring phosphoric acid to a specific aspartic acid site.. Phosphorylated PhoP can then regulate the expression of a series of genes downstream, including many genes associated with bacterial pathogenicity and virulence.

PhoP/PhoQ调节系统示意图

Project Procedure

a.Identification of phosphorylation sites

In order to identify other possible phosphorylation sites on the PhoP protein, the overexpressed PhoP was first purified by IP and then in-gel digested into peptides, and a tyrosine phosphorylation site on PhoP was identified by mass spectrometry analysis. This secondary mass spectrogram contained abundant fragment ions and the characteristic imine ion of tyrosine phosphorylation (216.043), identifying the phosphorylation site.

PhoP磷酸化位点的鉴定

b. Quantitative analysis of dimethylation

Dimethylation-based quantitative proteomics techniques were used to investigate what effect this tyrosine phosphorylation caused on the bacterial proteome. Typical process of quantitative proteomics: culture wild-type CmP strain, simulated phosphorylated YD mutant strain, simulated non-phosphorylated YF mutant strain, then extract the protein, enzymatically digest it into peptide fragments, double methylation markers are labeled as light,medium,heavy respectively, After mixing in equal proportions, perform mass spectrometry and quantitative analysis.

双甲基化定量分析

c. Results

In the two sets of biological replicates, about 1600 proteins were quantified, of which 200 were up-regulated and 274 were down-regulated. Correlation analysis showed good reproducibility between the two times.

Data overview

Extracting the quantitative information of the PhoP-activated proteins that have been reported in the literature and making such a heat map (below), it can be seen that they were all significantly down-regulated in the simulated-phosphorylated strain, whereas they were largely unchanged in the simulated-unphosphorylated YF strain.

已知的PhoP激活的蛋白

When examining the changes in PhoP and PhoQ at the protein level and mRNA level, it was found that PhoP was much more reduced than PhoQ at the protein level after the simulated-phosphorylated Y to D mutation, while the changes at the mRNA level were not significantly different.

The in vivo protein degradation experiments revealed that PhoP underwent protein degradation after the YD mutation. As can be seen in the figure below, only the YD mutation, which mimics phosphorylation, underwent degradation over time, while no degradation occurred for wild-type PhoP, the YF mutation, which mimics non-phosphorylation, and the D52A mutation, which also causes inactivation of PhoP protein.

Degradation experiment of protein in vivo

Functional PhoP is essential for the growth and survival of bacterial in the environment of antimicrobial peptides. From this experiment, it can be seen that the resistance of YD mutation mimicking phosphorylation and PhoP strain inactivating PhoP to antimicrobial peptides decreased significantly.

图抗病菌肽抵抗实验

d.Conclusion

Finally, a simple pattern diagram is presented as a summary of these findings above. When the classical D52 known on PhoP is phosphorylated by PhoQ, PhoP is activated and plays an important regulatory role. In contrast, when this Y site is phosphorylated by some kinase, PhoP is degraded away and thus loses its regulatory activity.

PhoP degradation mode diagram

　　1.Notices of samples delivery

2. General principles of proteomics sample preparation

a) Make sure the samples are in good condition and conform to the expected phenotypic or molecular indices.

b)Operate with gloves, mask and hood to avoid keratin contamination.

c) Ensure that operations after samopling are performed on ice and as quickly as possible.

d) According to the subsequent experimental needs, do the partitioning well to avoid repeated freezing and thawing of the protein.

e) After the material is taken, use liquid nitrogen to quick-freeze the material, store it in - 80 ℃ refrigerator, and send it with dry ice.

3.Sample volume requirements for proteomics service projects

Sample Category	Sample size
Silver-stained or Komas-stained glue strips	needs to be clearly visible
Cells	1 x 10^6 or >30 μL for proteomics; 1 x 10^7 for phosphorylated proteomics
Animal Tissue	100 mg fresh weight for proteomics items; 500 mg for phosphorylated proteomics items
Plant tissues	200 mg fresh weight for proteomics projects; 1 g for phosphorylated proteomics projects

Note: For samples with low protein yields, it is recommended to increase the sample delivery volume if possible; for specific samples, please contact the company's technical staff to negotiate the delivery volume.

4.Proteomics Service Project Sample delivery Guidelines

Sample Type	Sample Delivery Requirements
Glue strip samples	Protein strips are cut off with a clean blade and cut into 1 mm3 pieces and sent in imported EP tubes in time for sample delivery.
Cellular and bacterial samples	The samples should be washed three times with pre-cooled PBS buffer to remove the components of the culture medium. After washing, the supernatant should be discarded by centrifugation, snap frozen in liquid nitrogen and sent with dry ice.
Animal tissue samples	After retrieval, the tissue was quickly cut into 0.5 mm3 pieces, washed three times with pre-cooled PBS to remove residual blood, weighed, snap frozen in liquid nitrogen, and sent with dry ice.
Plant tissue samples	After sampling, the material is removed from the surface with pre-cooled PBS, weighed, snap frozen in liquid nitrogen, and sent with dry ice.
Protein solution samples	After determination of protein concentration, send frozen protein solution on dry ice (specify buffer composition and concentration); or precipitate protein by acetone method, dry, and send protein precipitate on dry ice or ice pack.
Culture supernatant	Cells are cultured to the appropriate density, washed three times with serum-free medium, added to serum-free medium and continued to incubate for the appropriate time, the culture supernatant is collected, centrifuged to remove the cells, filtered using a 0.22 μm membrane, snap-frozen in liquid nitrogen, and sent with dry ice.
Blood samples	Blood samples should be prepared as serum or plasma samples to avoid hemolysis. It is recommended to use the appropriate blood collection tubes and then obtain serum or plasma by centrifugation.
Phosphorylation samples	For phosphorylation analysis, samples are processed at low temperature and protease inhibitors and phosphatase inhibitors are added to the lysate.