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Protein qualitative analysis services
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Protein qualitative analysis services

Product Description
Case Analysis
Result display
Sample delivery
Q&A

a. Protein full-spectrum analysis

Protein full-spectrum analysis can also be called mass spectrometry shotgun analysis. which refers to the component analysis.The Objects of study are complete tissues, body fluids or their extracts, with the aim of identifying as many peptides and protein molecules as possible. The basic principle of protein mass spectrometry identification is to digest proteins into peptide mixtures by proteases, and then ionize them by electron bombardment or other means to form charged ions with different mass-to-charge ratios, and then separate the peptide ions with specific mass-to-charge ratios by a mass analyzer. The protein was identified by comparing the actual spectrogram with the primary and secondary mass spectra of protein digested by protease in theory.

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Schematic diagram of protein full spectrum analysis

 

Instruments used: Orbitrap Eclipse, Orbitrap Exploris 480, Q Exactive plus; AB SCIEX: TripleTOF 5600 plus.

Technical advantages: Comprehensive identification of a large number of proteins in whole cells, tissues or complex mixed samples, with 200-5000 proteins can be identified.

Application areas: Identification of all expressed proteins in tissues, cells or organelles in a given physiological state.

Sample requirements:

Sample

Sample requirements

Protein solution

Total protein > 500 μg, concentration > 0.1 μg/μL; Buffer solution without detergent NP40, Triton X-100, etc.

 

Lead time(Project Cycle)

Progress 

Time period

Sample collection/Quality control

2-3 working days

Sample processing 

4-5 working days

Mass spectrometry detection

2-3 working days

Data analysis

2-3 working days

Total 

No more than 15 working days

Note: For more than 5 samples, 3 working days will be added for each additional sample. If there is an urgent need, please contact us for negotiation.

 

b. Protein Identification

LC-MS/MS technology was used to identify proteins for moderately complex samples such as single strips (i.e., SDS-PAGE samples), IP, Co-IP, and Pull-down purification solutions.

凝胶电泳条带鉴定路线图

 

Pull-down experiment roadmap

 

Instruments: Orbitrap Eclipse, Orbitrap Exploris 480, Q Exactive Plus .

Technical advantages: high sensitivity, high accuracy. Suitable for moderately complex protein samples, such as IP, Co-IP .

Application areas: disease marker research, mechanism of action research, plant resistance research, drug target research, specific functional protein identification.

 

Sample requirements:

Sample

Sample requirements

SDS-PAGE bands  

Komas staining, silver staining (mass spectrometry compatible) bands are clearly visible

Protein solution Total protein >5μg, concentration >0.1μg/μL;

buffer without detergent NP40, Triton X-100, etc.

 

Lead time(Project Cycle)

Progress Time period
Sample collection/Quality control

1-2 working days

Sample processing 

2-3 working days

Mass spectrometry detection

2-3 working days

Data analysis     

2-3 working days

Report generation

1-2 working days

Total 

No more than 10 working days

Note: For more than 10 samples, 1 working days will be added for each additional sample. If there is an urgent need, please contact us for negotiation.

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.

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Schematic diagram of PhoP/PhoQ regulating system

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.

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图 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.

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Dimethylation quantitative analysis

 

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.

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数据概述

 

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.

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Figure Known PhoP-activated protein

 

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.

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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.

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Antimicrobial peptide resistance test

 

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.

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PhoP degradation mode diagram

持续更新中,敬请期待...

Notices of samples delivery

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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 specification
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 EP tubes and send the sample in time. When sending the sample, add ice bags to maintain the low temperature

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 with dry ice (specify buffer composition and concentration); or precipitate protein by acetone method, dry, and send protein precipitate with 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.

Stay tuned for further updates...

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