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Full-ion monitoring and quantifying method based on second-level mass spectrum

A secondary mass spectrometry and ion monitoring technology, applied in the field of all ion monitoring and quantification based on secondary mass spectrometry, can solve the problems of loss of quantitative information of secondary spectrum, affecting the quantitative sensitivity of low-abundance proteins, reducing the proteome, etc., to achieve quantitative Serious information loss, easy to be disturbed by background noise, and high detection sensitivity

Active Publication Date: 2017-01-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

In the proteome analysis based on the shotgun method, the current mass spectrometry data acquisition mainly adopts the data-dependent mode, in which the peptide precursor ions of low-abundance proteins are easily suppressed by the high-abundance peptide precursor ions, and the acquisition of MS / MS is impossible , thus losing the qualitative and quantitative information of low-abundance proteins, reducing the sensitivity and accuracy of protein qualitative and quantitative analysis
At the same time, although the setting of dynamic exclusion can significantly increase the number of identified proteins, most peptides can only trigger one collision fragmentation, so only the secondary spectrum ions of the peptide at a specific moment can be collected for quantitative analysis. Reduced the sensitivity and accuracy of proteomics, especially the quantification of low-abundance proteins (Zhang, Y.Y., et al. (2013). "Protein Analysis by Shotgun / Bottom-up Proteomics." Chemical Reviews 113(4):2343- 2394)
In addition, the introduction of more than 4 deuterium labels will cause shifts in the retention time of light and heavy labeled peptides, and the setting of dynamic exclusion will lead to a decrease in quantitative accuracy
In recent years, the developed quantitative methods (SWATH18, PRM19) based on data-independent mode mass spectrometry acquisition can effectively solve the problem of loss of quantitative information in secondary spectra, but the complexity of the generated secondary mass spectra has doubled (Rost , H.L., et al.(2014). "OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data." Nature Biotechnology 32(3):219-223.)
In addition, this method cannot realize the simultaneous qualitative and quantitative analysis of proteins in a single experiment, and background noise seriously interferes with the fragment ions of low-abundance peptides, which affects the quantitative sensitivity of low-abundance proteins

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0014] 1. Protein enzymatic hydrolysis: Mix two copies of Hela cells cultured by SILAC kit and respectively labeled with light and heavy lysine at a ratio of 1:1, and use 8mol / L urea solution (dissolved in 50mM ammonium bicarbonate solution, pH 8 ) after the protein was ultrasonically crushed and extracted, the protein concentration was measured using a BCA kit, and the measured protein concentration was 1 mg / mL. Take 1 mL of protein solution with a concentration of 1 mg / mL, add 10 μL of 10 mM dithiothreitol (DTT) solution, and react at 56° C. for 2 h to denature and reduce the protein. Subsequently, 10 μL of 25 mM iodoacetamide (IAA) solution was passed through, and reacted in the dark at room temperature for 30 min to carry out the alkylation treatment of the protein. Finally, 25 μg of trypsin (dissolved in 50 mM ammonium bicarbonate solution, pH 8) was added for enzymatic hydrolysis of the protein. After 12 hours of enzymatic hydrolysis at 37°C, 10 μL of formic acid soluti...

Embodiment 2

[0019] 1. Protein enzymatic hydrolysis: Mix two copies of mouse liver cancer high and low metastatic cells cultured by SILAC kit and respectively labeled with light and heavy lysine at 1:1, and use 8mol / L urea solution (dissolved in 50mM bicarbonate ammonium solution, pH 8) to ultrasonically extract the protein, and then use the BCA kit to measure the protein concentration, and the determined protein concentration is 1 mg / mL. Take 1 mL of protein solution with a concentration of 1 mg / mL, add 10 μL of 10 mM dithiothreitol (DTT) solution, and react at 56° C. for 2 h to denature and reduce the protein. Subsequently, 10 μL of 25 mM iodoacetamide (IAA) solution was passed through, and reacted in the dark at room temperature for 30 min to carry out the alkylation treatment of the protein. Finally, 25 μg of trypsin (dissolved in 50 mM ammonium bicarbonate solution, pH 8) was added for enzymatic hydrolysis of the protein. After 12 hours of enzymatic hydrolysis at 37°C, 10 μL of formi...

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Abstract

The invention relates to a full-ion monitoring and quantifying method based on a second-level mass spectrum. The method comprises a mass spectrum data collection step and a mass spectrum data processing step, wherein the mass spectrum data collection step comprises the step of combining a peptide fragment liquid phase classification technique with a mass spectrum gas phase classification technique, and collecting mass spectrum data by virtue of a non-dynamic exclusion type data dependence mass spectrum scanning mode, so as to realize full collection of mass spectra of fragment ions of all peptide fragments; and the mass spectrum data processing step comprises the step of after the collection of the mass spectrum data, making comparison on intensities of mass spectrum peaks of second-level spectrum ions of multiple labeled peptide fragments of the same peptide fragment, so as to realize the quantitative analysis of proteins among different samples. The method has the advantages of high detection sensitivity, low noise disturbance, good repeatability and high quantifying accuracy and further has wide application prospects in the large-scale qualitative and quantitative analysis of proteomics.

Description

technical field [0001] The invention relates to a quantitative method for all ion monitoring based on secondary mass spectrometry and its application, which can realize high-accuracy quantification of protein samples, has the advantages of high detection sensitivity, low noise interference, and good reproducibility. Large-scale qualitative and quantitative analysis has broad application prospects. Background technique [0002] In recent years, with the development of high-precision biological mass spectrometry technology and the progress of massive data processing technology based on bioinformatics, proteome quantitative methods based on biological mass spectrometry have become the mainstream technology of quantitative proteome. In the proteome analysis based on the shotgun method, the current mass spectrometry data acquisition mainly adopts the data-dependent mode, in which the peptide precursor ions of low-abundance proteins are easily suppressed by the high-abundance pept...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/72
Inventor 张丽华赵群单亦初张珅杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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