Protein Quantification Service_Wuhan biological Optical Valley Science and technology

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Protein Quantification Service

Product Description

Case Analysis

Result display

Sample delivery

Q&A

a.TMT™ Technology

TMT™ (Tandem Mass Tag™) technology is an in vitro peptide labeling technology developed by Thermo Scientific, which is designed for the identification and quantification of proteins in different samples by tandem mass spectrometry (MS). This technique uses multiple isotope labels to label the amino groups of peptides, and after LC-MS/MS analysis, the relative amounts of proteins in 2-16 groups of different samples can be compared simultaneously.

TMT ™ Technical schematic diagram

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

Technical advantages: Simultaneous analysis of protein differences in 2-16 samples of different conditions. Suitable for a wide range of sample types: cytoplasmic proteins, membrane proteins, nuclear proteins, extracellular proteins, etc. Compared with protein quantification by gel separation, TMT mass spectrometry can detect more types of protein molecules, such as low-abundance proteins, strongly acidic (base) proteins, <10KD or >100KD protein molecules.

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

Sample requirements

Sample Type	Sample requirements (per group of samples)
Protein extract	Concentration >1μg/μL, total protein >300μg
Cell sample	Cell volume >10^7
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	5-6 working days
Mass spectrometry test	3-4 working days
Data analysis	2-3 working days
Total	No more than 20 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.

b.Label-free

Label-free quantification is the mass spectrometric analysis of enzymatic protein peptides by liquid chromatography-mass spectrometry (LC-MS).

Label-free quantitative proteomics experimental process

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

Technical advantages: no labeling of proteins, low cost and low volume of samples required.

Application areas: disease marker screening, mechanism of action research, plant resistance research, drug target research, special function protein screening.

c.SWATH/DIA

SWATH/DIA is a new panoramic scanning mode for mass spectrometry. Compared with the traditional shot-gun technique, SWATH/DIA acquisition mode is able to scan all precusor peptide in the scan interval at ultra-high speed and perform secondary fragmentation, thus obtaining complete peptide information. With SWATH /DIA acquisition mode, complete quantitative and qualitative results can be obtained in a single experiment, without method optimization. SWATH/DIA mode overcomes the problem of shot-gun scan randomness, resulting in better data reproducibility. SWATH/DIA is particularly suitable for enrichment of purified samples, e.g. SWATH/DIA quantification after IP to detect proteins that interact with the target protein.

SWATH technical schematic diagram

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

Technical advantages

High sensitivity, which has the data acquisition mode of MRM, combined with a high-resolution mass spectrometry system with comparable sensitivity to MRM.

Quantitative correlation between duplicate samples can reach above 0.99.

Quantitative accuracy almost comparable to MRM technique.

the quantification range can span 4 orders of magnitude

The quantitative results are very good.

Application areas: mechanism and regulatory mechanism research, screening of disease markers, screening of drug use and prognostic markers, molecular typing of diseases.

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 day
Sample processing	5-6 working days
Mass spectrometry test	3-4 working days
Data analysis	2-3 working days
Total	No more than 20 working days

Note: For more than 4 samples, 5 working day 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.

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.

Figure Identification of PhoP phosphorylation site

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.

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.

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.

Known PhoP-activated proteins

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.

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.

PhoP degradation mode diagram

1. Proteomics project service process

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
细胞、细菌样品	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.