SILAC-based mass spectrum method for absolute quantification of serum cystatin C

An absolute quantitative and cystatin technology, applied in the field of mass spectrometry detection, can solve problems such as inaccurate quantitative results, achieve the effect of improving sensitivity, anti-interference ability, and high extraction efficiency

Pending Publication Date: 2022-07-05
GUANGDONG HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Methods for quantifying proteins / peptides based on ID / MS are divided into four categories, each with its own advantages and disadvantages: AQUA method is the most widely used, because AQUA method can not only be used for absolute quantification of conventional proteins, but also for post-translational modifications (such as phosphorylation) Absolute quantification of protein, and isotope-labeled peptides can be purchased commercially, which is very convenient to use, so this method is widely used in absolute quantification of proteins, but the internal standard peptide is added after proteolysis, ignoring the sample pretreatment process Errors caused by the loss of target protein and incomplete enzymatic hydrolysis, so the quantitative results are not accurate
However, there is still no method for absolute protein quantification based on the SILAC method using intact protein as an isotope-labeled internal standard for serum Cys C

Method used

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  • SILAC-based mass spectrum method for absolute quantification of serum cystatin C
  • SILAC-based mass spectrum method for absolute quantification of serum cystatin C
  • SILAC-based mass spectrum method for absolute quantification of serum cystatin C

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 115

[0033] Example 1 15 Prokaryotic expression and protein purification of recombinant cystatin C labeled with N isotope

[0034] 1) Target gene synthesis

[0035] The amino acid sequence of Cys C protein was retrieved from the Pubmed protein library, and the sequence was optimized. The amino acids 1-20 of the analyzed protein are the transmembrane region, then the signal peptide is removed, and a 6xHis tag is fused to the carboxyl terminus of the sequence. The purpose is to facilitate the purification of proteins using nickel columns in subsequent experiments. The corresponding sequence was added during optimization, and the molecular weight of the target protein was about 15kD after optimization. The corresponding nucleic acid sequences were obtained in the Pubmed gene bank. For the sequence analysis, designs such as codon usage preference adjustment, codon usage distribution optimization and GC content optimization were performed in PrimerPrimer 5.0 software. Analysis of r...

Embodiment 2

[0075] Example 2 Selection of Cys C Featured Peptides

[0076] (1) Cys C protein sequence information

[0077] The Cys C protein sequence information from the NCBI protein database is as follows:

[0078] Cystatin-C precursor[Homo sapiens]

[0079] NCBI Reference Sequence: NP_000090.1

[0080] The protein sequence is as follows:

[0081] 1 magplrapllllailavalavspaagsspgkpprlvggpmdasveeegvrraldfavgey

[0082] 61 nkasndmyhsralqvvrarkqivagvnyfldvelgrttctktqpnldncpfhdqphlkrk

[0083] 121 afcsfqiyavpwqgtmtlskstcqda (SEQ ID NO: 1)

[0084] (2) Prediction of peptides produced by Cys C hydrolysis by trypsin.

[0085] The PeptideMass tool in the ExPASy software predicts the peptide segment produced by Cys C hydrolysis by trypsin, the theoretical isoelectric point pI: 8.75 / Mw (average mass), and the molecular weight: 13347.14.

[0086] (3) Retrieval and analysis of physicochemical properties of characteristic peptides.

[0087] The physicochemical properties of the characteristi...

Embodiment 3

[0089] Example 3 Determination of Mass Spectrometry Conditions

[0090] Confirmation of peptide standard mass spectrometry conditions: For the screened characteristic peptides, the biological company will synthesize the characteristic peptides and their isotope-labeled internal standards. Prepare 1 mL of standard peptides with a concentration of 1 μg / mL, and inject by needle pump.

[0091] Cys C protein standard and 15 N-labeled Cys C protein standard mass spectrometry confirmation: 10 μg Cys C or 15 The N-labeled Cys C protein standard was subjected to protein denaturation, reductive alkylation and proteolysis according to the protein sample pretreatment steps, and then injected by needle pump.

[0092] (1) Ion pair selection

[0093] 1) Q1 MS first-level full scan.

[0094] 2) Characteristic peptide product ion scan (Product Ion MS 2)

[0095] Enter the mass-to-charge ratio of the precursor ion;

[0096] Optimize CE, range 5-30, observe the signal intensity of product ...

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Abstract

The invention relates to the technical field of mass spectrum detection, in particular to a mass spectrum method for absolute quantification of serum cystatin C based on SILAC. The invention discloses establishment and performance evaluation of an absolute quantification method for human serum cystatin C based on SILAC, the absolute quantification of a target protein is carried out by using a complete CysC protein marked by 15N as an internal standard for the first time, and result errors caused by sample loss, incomplete enzyme digestion and the like in a sample pretreatment process are greatly reduced. Specific anti-M270 epoxy resin magnetic bead co-immunoprecipitation is adopted in sample pretreatment, the extraction efficiency is higher than that of an existing method, and the actual sample detection sensitivity and the anti-interference capacity are improved. According to the invention, a CysC national standard substance GBW09870 is combined with an amino acid analysis method to carry out quantity value traceability of CysC detection in serum for the first time.

Description

technical field [0001] The invention relates to the technical field of mass spectrometry detection, in particular to a mass spectrometry method for absolute quantification of serum cystatin C based on SILAC. Background technique [0002] Serum cystatin C (Cys C), as a new type of endogenous GFR substance, is not affected by the patient's age, gender, body weight, height, bilirubin, blood sugar, and nutritional status. An ideal and reliable endogenous marker for changes in GFR. The concentration of Cys C in serum is low, and the assay method requires high analytical sensitivity and specificity. Early detection methods of Cys C content in serum include enzyme immunoassay (EIA) and one-way immunodiffusion (RID), but the sensitivity of these methods is poor. Subsequently, there are simpler and more sensitive fluorescence immunoassay (FIA) and radioimmunoassay (RIA) methods to improve the reliability of Cys C content determination. These assay methods are all heterogeneous dete...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/539G01N33/58G01N33/68G01N30/02G01N30/06G01N30/72G01N27/64
CPCG01N33/539G01N33/581G01N33/6851G01N33/6842G01N30/02G01N30/72G01N30/06G01N27/64G01N2333/8139G01N2030/045G01N2030/062
Inventor 黄宪章张乔轩白开严君韩丽乔林海标展敏张鹏伟王建兵柯培锋庄俊华
Owner GUANGDONG HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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