Method for fast enriching and appraising phosphopeptide on MALDI-TOF-MS sample target

A technology for enriching phosphopeptides and phosphopeptides, applied in the field of analytical chemistry, can solve the problems of large-scale proteomics, high-throughput analysis, and sample loss, and achieve the effects of convenient transformation, low cost, and loss prevention

Inactive Publication Date: 2008-06-11
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the manual operation in the centrifuge tube, the transfer process leads to sample loss, and only a few samples can be processed at a time, which cannot meet the requirements of large-scale and high-throughput analysis of proteomics

Method used

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  • Method for fast enriching and appraising phosphopeptide on MALDI-TOF-MS sample target
  • Method for fast enriching and appraising phosphopeptide on MALDI-TOF-MS sample target
  • Method for fast enriching and appraising phosphopeptide on MALDI-TOF-MS sample target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Take a certain amount of prepared nanoparticles into a 1.5 mL centrifuge tube, add 0.15% trifluoroacetic acid solution, and sonicate for 1 minute to obtain a 0.3 mg / mL nanoparticle suspension.

Embodiment 2

[0034]Pipette 0.8 μL (240 ng) of nano-ion suspension and distribute it on the sample hole of the stainless steel target of matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (4700 proteomics Analyzer), and load 2 pmol of α-casein hydrolyzate after drying , incubate at room temperature for 10 minutes; immerse the target in a petri dish filled with 40 mL of 20% acetonitrile-0.1% trifluoroacetic acid solution, wash gently on a shaker for 15 minutes to remove non-phosphopeptides and salts, take out and rinse the target once with Millpore water , add DHB matrix (20mg / mL-DHB-%H 3 PO 4 -50% ACN) co-crystallized; MALDI-TOF-MS analysis was performed. When not enriched, there are many non-phosphopeptides, and only 3 phosphopeptides are detected, such as figure 2 a, While most of the non-phosphopeptides were removed after the enrichment of the modified MALDI target, nine phosphopeptides were detected, as figure 2 as shown in b. Detected phosphopeptides are...

Embodiment 3

[0036] Pipette 0.8 μL of nano-ion suspension and distribute it on the sample hole of the stainless steel target of matrix-assisted laser desorption / ionization time-of-flight mass spectrometer, load 2 pmol of β-casein hydrolyzate after drying, and repeat the operation of Example 2 to enrich Collection, MALDI-TOF-MS analysis. When not enriched, only one phosphopeptide was detected, and the signal-to-noise ratio was very low. After enrichment, most of the non-phosphopeptides disappeared, and two phosphopeptides were detected, which became the two strongest peaks in the spectrum. Such as image 3 shown in a-b.

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Abstract

The invention relates to a method of fast enrichment and fast identification on the phosphoeptide of the modificatory sample target by the matrix-assisted laser-desorption / time-of-flight mass spectrometry (MALDI-TOF-MS), which is to physically adsorb the fine magnetic nano particle onto the sample target, wash the sample after the sample is loaded to remove the non phosphoeptide and phosphate, add the matrix to make cocrystallization with the phosphoeptide absorbed on the sample target and then conduct the MALDI-TOF-MS analysis. The method does not relate to the chemical reaction without pre-processing to the target, which can modify the target MALDI made of different substrate materials; A plurality of samples complete their enrichment at the same time on the modified target MALDI; There are no sample transfer in the whole process from the sample loading to the mass spectrometric analysis. The invention is easy, fast and sensitive to operate, which has a good application value in the large-scale analysis of the phosphoproteome.

Description

technical field [0001] The invention belongs to the field of analytical chemistry and relates to a method for enriching and identifying phosphopeptides. Specifically, it is a method for rapidly enriching and identifying phosphopeptides on a modified matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS) sample target. Background technique [0002] Phosphorylation of protein is one of the important covalent modifications in organisms, and it plays an important role in the regulation of cell proliferation, differentiation, signal transduction, apoptosis and other life processes. In recent years, mass spectrometry (MS) has been widely used for the identification of phosphorylated proteins. Due to the low stoichiometry of phosphorylated proteins in cells / tissues and the signal suppression of phosphopeptides by non-phosphorylated peptides during mass spectrometry analysis, direct mass spectrometry analysis of phosphorylated proteins or phosph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N30/08G01N30/60
Inventor 钱小红谭峰张养军王京兰蔡耘应万涛
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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