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Multi-dimensional dual-channel liquid chromatography-mass spectrometry device

A liquid chromatography and mass spectrometry technology, applied in the field of liquid chromatography-mass spectrometry, can solve the problems of sample loss, sample loss, and unsatisfactory orthogonality, etc., to reduce sample loss, prolong service life, and reduce The effect of the risk of contamination

Active Publication Date: 2022-04-19
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, both of these two representative methods have inherent defects: the method of desalting through a C18 trapping column generally uses a part of C18 as a two-phase trapping column downstream of the SCX, which requires disconnecting the flow path of the liquid phase so that the The organic mobile phase gradient can be used to elute the peptides retained in the analytical column after the salt composition is completely flushed into the waste solution; while for the pH gradient method, although it can use the traditional MudPIT setup, it needs to be used in the SCX column. Gradients instead of salt gradients, or the addition of new loop-trap columns to complete the acid-salt-pH gradient of MudPIT, such orthogonality is not satisfactory or the dead volume is too high, resulting in a large amount of sample loss, and these The method is too complex to be widely used
In order to achieve in-depth research on proteomics without polluting the mass spectrometer, people have recently tried to conduct offline operation, however, the offline mode is not only inefficient but also causes a large amount of sample loss

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1-preliminary preparation of samples

[0047]1. Cell culture: Take out the frozen A549 cells in liquid nitrogen, shake and thaw in a water bath at 37°C, remove the supernatant by centrifugation, add 1ml of fresh medium by blowing and mixing, transfer to a culture bottle, add 9ml of fresh medium and mix well, put in Incubate at 37°C for 48 hours until the cells cover the bottom flask wall.

[0048] 2. Cell lysis: After the cells cover the bottom bottle wall, take the cells out of the incubator, discard the culture medium, wash twice with PBS, add 2 mL of trypsin to each bottle of cells for 2 minutes, and collect the cells for lysis solution into a 15mL centrifuge tube, centrifuge to remove the supernatant, and wash three times with PBS.

[0049] 3. Protein extraction: add 1% protease inhibitor (v:v) to RIPA lysate, every 10 6 Add 1ml RIPA lysate to each cell, lyse on an ice shaker for 30 minutes, and blow with a pipette every 10 minutes. Centrifuge at 14000r...

Embodiment 2

[0052] Preparation and sample loading of embodiment 2-chromatographic column

[0053] 1. Preparation of capillary with tip: Take a quartz capillary with a length of about 55 cm, burn off the outer layer of polyimide in the middle part of the capillary with the outer flame of an alcohol lamp, use a wiping paper to dip in methanol to wipe clean the burnt part, and place it in A capillary containing a tip is prepared in a laser micropipette needle puller for use.

[0054] 2. Preparation of capillary containing Kasil plug: select 15cm-20cm long, 250μm inner diameter undeactivated (undeactivated) quartz capillary, potassium silicate solution Mix evenly with ammonium formate solution according to 3:1 (v / v), prepare it now, use the capillary phenomenon to make the mixed solution enter one end of the quartz capillary, and then place it in an oven at 100°C for 4 hours, so that the mixed solution entering the capillary becomes porous and more Polymer. Cut the plug to a length of 0....

Embodiment 3

[0057] Embodiment 3-LC-MS / MS analysis

[0058] 3.1 The device setup of the classic MudPIT and its liquid phase setup are as follows.

[0059] (1) The device setting of classic MudPIT: the device of classic MudPIT is as follows figure 1 Shown: One end of the PEEK miniature tee is connected to the liquid chromatography pump, the other end is connected to the SCX chromatographic column, and one end of the vertical flow path is connected to the liquid junction voltage device of the mass spectrometer. The liquid junction voltage device is a device that applies a voltage to the mobile phase at the initial end of the chromatographic column through a metal electrode to charge the analyte. The SCX column is then connected to the analytical column via a PEEK micro union.

[0060] (2) Liquid phase setup of classic MudPIT: The liquid phase used in this experiment is Thermo EASY-nLC TM The 1200 liquid phase device, by placing ammonium acetate of different concentrations on the corresp...

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Abstract

The invention relates to a multi-dimensional dual-channel liquid chromatography-mass spectrometry device, which includes a first liquid phase pump, a second liquid phase pump, a six-way valve, a primary analysis column, a first secondary analysis column, a second Components for liquid chromatography with stage analytical columns and analytical column position switching devices. The liquid chromatography-mass spectrometry device of the present invention can prevent the damage to the mass spectrometry system caused by the specific mobile phase components in the multidimensional liquid chromatography, and has a good effect especially in the research of proteomics.

Description

technical field [0001] The invention belongs to the technical field of liquid chromatography-mass spectrometry (liquid chromatography-mass spectrometry) in chemical analysis, and in particular relates to a multi-dimensional dual-channel liquid-mass spectrometry device and an application method of the device, especially in proteomics applications in identification. Background technique [0002] Protein is the translation product of the genetic information of living organisms and the main bearer of biological functions, catalyzing and controlling most of the processes in living organisms. Traditional protein research mainly focuses on the structure and function of a single protein, and matches the results with the existing gene sequence information to explain the physiological or pathological phenomena of the body. However, due to the complexity of life activities, most physiological or pathological processes often require the participation of multiple biomolecules (especiall...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/20
CPCG01N30/20G01N2030/201
Inventor 黄超兰高帅鑫张楠
Owner PEKING UNIV
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