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Method for carrying out rapid peptide diagram analysis on protein

A protein peptide and protein technology, applied in the field of drug analysis, can solve the problems of low resolution, limitation, and inability to realize comprehensive collection of sample information, so as to improve the analysis throughput and ensure accurate qualitative results

Pending Publication Date: 2022-03-04
BEIJING UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, Liu Yang et al. achieved the quantitative analysis of 13 amino acids through a targeted analysis strategy combining DI and MRM acquisition modes. However, this method only targeted the acquisition of individual amino acids of interest, and could not achieve comprehensive collection of sample information. The low-resolution QQQ mass spectrometer has a resolution of only 0.6-0.8Da, which is very limited in qualitative analysis
At present, there is no report on the application of DI-MS / MS combined with gaseous segmentation technology to the analysis of protein and peptide samples

Method used

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  • Method for carrying out rapid peptide diagram analysis on protein
  • Method for carrying out rapid peptide diagram analysis on protein
  • Method for carrying out rapid peptide diagram analysis on protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of embodiment 1 experimental material and sample

[0029] 1.1 Experimental materials

[0030]Standard protein human serum albumin (human serum albumin, HSA, Shanghai Yuanye Biotechnology Co., Ltd.); trichloroethyl phosphate (TCEP, Beijing Biotuoda Technology Co., Ltd.); iodoacetamide (IAA, Sigma Corporation, USA ); Urea (Beijing Chemical Plant); Ammonium Bicarbonate (NH 4 HCO 3 , Beijing Chemical Plant); trypsin (Promega, USA). Mass spectrometry grade acetonitrile (ACN) and formic acid (FA) were purchased from Thermo Fisher, USA, and deionized water was Milli-Q ultrapure water (18.2 MΩ·cm) made in the laboratory.

[0031] METTLER XS105 electronic analytical balance (Mettler-Toledo Instrument Co., Ltd.); 10kDa ultrafiltration tube (Sartorius); MQD-S2P constant temperature double-layer shaker shaker (Shanghai Minquan Instrument Co., Ltd.).

[0032] 1.2 Sample preparation

[0033] Sample preparation strategies in the prior art [ JR,Zougman A,Nagara...

Embodiment 2

[0037] Embodiment 2 DI-MS / MS combined with gaseous segmentation technology and LC-MS / MS (control group) determination

[0038] 2.1 Determination by DI-MS / MS combined with gaseous segmentation technique

[0039] Peptide samples are directly introduced into SCIEX TripleTOF 6600 without separation by chromatographic columns + In the ESI ion source equipped with the mass spectrometer. The automatic sampling of the sample is realized by the LC system at the front end of the MS, and the specific operation is as follows: the mobile phase pump transmits the aqueous solution (A) containing 0.1% FA and the acetonitrile solution containing 0.1% FA according to the ratio of 50:50 (v / v) ( B), the flow rate gradient is: 0~0.5min, 50μL / min; 0.5~0.51min, 50~7μL / min; 0.51~4.5min, 7μL / min; 4.5~4.51min, 7~50μL / min; 4.51~5min , 50 μL / min. In the first stage (0-0.5min), the sample is quickly transferred to the ion source through a mobile phase with a large flow rate; in the second stage (0.5-...

Embodiment 3

[0045] Embodiment 3 experimental result

[0046] 3.1 Peptide Characterization by DI-MS / MS Combined with Gas Segmentation Technology

[0047] The determination of DI-MS / MS combined with gaseous fractionation technology includes MS 1 MS in full scan and unit mass window 2 Atlas. To improve MS 1 Data Quality, MS 1 The spectral acquisition time was set to 10s. MS 1 The main signals in the spectrum appear at m / z467.2622, 480.7839, 507.3021, 569.7515, 673.3769, 772.4380, 789.4710 and 1013.5991, such as figure 1 As shown in A. Further we analyzed each MS 1 The charged number of the signal is of great significance for molecular formula prediction and amino acid sequence identification. Using natural abundance of 1.01% 13 The C-isotope signal to confirm the charged state is manifested as a correlation signal with 13 If the distance between the C-isotope signals is 1.000Da, then the ion is a singly charged ion; if the distance is 0.500Da, it corresponds to a doubly charged...

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Abstract

The invention provides a method for carrying out rapid peptide diagram analysis on protein, the method adopts DI-MS / MS combined with a gaseous segmentation technology and can be suitable for all protein drugs, theoretical enzymolysis peptide fragments, precursor ions and fragment ion information obtained by computer assistance are matched with peptide diagrams collected by the DI-MS / MS combined with the gaseous segmentation technology, and the peptide diagrams obtained by the DI-MS / MS combined with the gaseous segmentation technology are subjected to rapid peptide diagram analysis. The quality analysis and control of all protein drugs can be realized. According to the method, only 5 min is needed for single analysis, compared with traditional LC-MS technology-based polypeptide sample analysis, the analysis flux is greatly improved, more accurate m / z value information can be provided, the accuracy can reach four or more decimal points, accurate qualification of protein is guaranteed, and the method has a wide application prospect.

Description

technical field [0001] The invention relates to the field of pharmaceutical analysis, and more specifically, the invention relates to a method for rapid peptide map analysis of proteins by using DI-MS / MS combined with gaseous segmentation technology. Background technique [0002] In recent years, protein drugs such as monoclonal antibodies (mAbs), immunoglobulins, and interferons have been widely used, but their quality control is limited by the lack of suitable analytical methods and platforms. Compared with small molecule drugs, the quality control and analysis of protein drugs is still a huge challenge due to the lack of suitable analytical methods and the difficulty of obtaining standard proteins. MALDI-TOF-MS high-resolution mass spectrometry can measure the accurate molecular weight of a specific protein without proteolysis, however, this technique cannot provide the required amino acid sequence information. The peptide map analysis strategy takes a series of peptide ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/72G01N27/62G01N33/68
CPCG01N30/02G01N30/72G01N27/62G01N33/6848
Inventor 宋月林李军张珂屠鹏飞龚兴成
Owner BEIJING UNIV OF CHINESE MEDICINE