Method and device for protein sequence analysis

A protein sequence and analysis method technology, applied in the field of protein sequence analysis, to achieve the effect of simple protein digestion and high sequence coverage

Pending Publication Date: 2021-08-06
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, few studies have addressed the application of microdroplets in biochemical analysis

Method used

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  • Method and device for protein sequence analysis
  • Method and device for protein sequence analysis
  • Method and device for protein sequence analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Optimizing Droplet-MS Performance.

[0086] To optimize the performance of droplet-MS, trypsinized ACTH was used as a simple model system.

[0087] 5 mM ammonium bicarbonate (NH 4 HCO 3 , pH 8) aqueous solution sample was injected into a self-made nebulizer (the inner diameter of the capillary was 50 μm, the outer diameter was 148 μm), thereby generating a micro-droplet flow (such as figure 1 shown).

[0088] Dry N at a pressure of 120 psi 2 With the aid of nebulizing gas, the sample solution was sprayed from the tip of a fused quartz capillary (outer diameter 148 μm, inner diameter 50 μm, Polymicro Technologies, China). Droplets were directed into MS for real-time detection by placing the nebulizer at an appropriate location in front of a high-resolution mass spectrometer (LTQ Orbitrap Elite, Thermo Scientific, San Jose, CA). The MS inlet capillary was kept at 275°C and the capillary voltage was kept at 0V. No other source of gas is used when digestion is perform...

Embodiment 2

[0099] Digestion and analysis of proteins that are particularly difficult to digest with trypsin using Microdroplet-MS.

[0100] Add myoglobin (10 μM) and trypsin (5 μg / mL) in 5 mM ammonium bicarbonate (NH 4 HCO 3 , pH 8) aqueous solution sample was injected into a self-made nebulizer (the inner diameter of the capillary was 50 μm, the outer diameter was 148 μm), thereby generating a micro-droplet flow (such as figure 1 shown).

[0101] Dry N at a pressure of 120 psi 2 With the aid of nebulizing gas, the sample solution was sprayed from the tip of a fused quartz capillary (outer diameter 148 μm, inner diameter 50 μm, Polymicro Technologies, China). Droplets were introduced into the MS by placing the nebulizer in an appropriate position in front of a high-resolution mass spectrometer (LTQ Orbitrap Elite, Thermo Scientific, San Jose, CA) and applying a +3 kV positive high voltage (BOHER HV, Genvolt, UK) to the nebulizer. Perform real-time detection. The MS inlet capillary w...

Embodiment 3

[0113] Digestion and analysis of cytochrome c by microdroplet-MS.

[0114] Following the same method as described in Example 1 and Example 2, the droplet-MS was further used for the digestion and analysis of cytochrome c at a positive voltage of +3 kV.

[0115] Such as Figure 8 As shown, 33 peptides of cytochrome c were successfully identified (corresponding to 83% sequence coverage). The results demonstrate that droplet-MS is a versatile tool for protein digestion.

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Abstract

The invention relates to a method and a device for protein sequence analysis, and the use of microdroplets for improving protein sequencing by accelerating enzymatic digestion. The method comprises the following steps: a) forming a solution containing protein into microdroplets having a size small enough to result in acceleration of protein digestion; b) introducing the microdroplets into a mass spectrometer (MS) for real-time detection; c) obtaining analysis result of the protein from the mass spectrometer (MS), wherein the protein is fully digested in the microdroplets before entering the mass spectrometer (MS). The method and device can achieve simple and nearly complete protein digestion in a very short time, and obtain higher sequence coverage.

Description

technical field [0001] The present invention relates to methods and devices for protein sequence analysis. More specifically, the present invention relates to a protein sequence analysis method and a droplet-MS (droplet-mass spectrometer) device for protein sequence analysis. The present invention also relates to the use of microdroplets for improving protein sequencing by accelerating enzymatic digestion, wherein the microdroplets are formed from a solution containing a protein and the size of the microdroplets is small enough to result in an acceleration of protein digestion. Background technique [0002] In bottom-up proteomics, enzymatic digestion of proteins is a key method essential to breaking down proteins into smaller polypeptides before analyzing their structure by mass spectrometry (MS) 1 . In a typical enzymatic digestion, a protein solution is mixed with an appropriate amount of enzyme (eg, trypsin) and incubated overnight at 37°C. However, such a process is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6818G01N33/6848G01N33/6821
Inventor R·N·扎尔仲小琴
Owner FUDAN UNIV
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