Strategies to verify equimolar peptide release in mass spectrometry-based protein quantification exemplified for apolipoprotein(a).

IF 3.8 2区 医学 Q1 MEDICAL LABORATORY TECHNOLOGY Clinical chemistry and laboratory medicine Pub Date : 2024-10-28 DOI:10.1515/cclm-2024-0539
Yuri E M van der Burgt, Fred P H T M Romijn, Maxim M Treep, L Renee Ruhaak, Christa M Cobbaert
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Abstract

Objectives: Quantitative protein mass spectrometry (MS) is ideally suited for precision diagnostics and for reference standardization of protein analytes. At the Leiden Apolipoprotein Reference Laboratory we apply MS strategies to obtain detailed insight into the protein-to-peptide conversion in order to verify that quantifier peptides are not partly concealed in miscleaved protein backbone.

Methods: Apolipoprotein(a) (apo(a)) was digested in a non-optimal manner to enhance the number of miscleaved peptides that were identified by high resolution liquid chromatography tandem-MS measurements. The protein-to-peptide conversion was carefully mapped with specific attention for miscleaved peptides that contain an apo(a) quantifier peptide. Four different isotopologues of each apo(a)-quantifier peptide were applied to evaluate linearity of internal peptide standards during measurement of specific real-life samples.

Results: Two apo(a) quantifier peptides that were concealed in two different miscleaved peptides were included into a multiple reaction monitoring list in our targeted MS-based apo(a) quantifications to alert for potential protein digestion discrepancies. The presence of miscleaved peptides could be ruled out when applying our candidate reference measurement procedure (RMP) for apo(a) quantification.

Conclusions: These data further corroborate the validity of our apo(a) candidate RMP as higher order method for certification of commercial Lp(a) tests that is endorsed by the International Federation of Clinical Chemistry and Laboratory Medicine. MS-based molecular detection and quantification of heterogeneous apo(a) proteoforms will allow manufacturers' transitioning from confounded lipoprotein(a) [Lp(a)] mass levels into accurate molar apo(a) levels.

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在基于质谱的蛋白质定量中验证等摩尔肽释放的策略,以脂蛋白(a)为例。
目的:定量蛋白质质谱法(MS)是精确诊断和蛋白质分析物参考标准化的理想方法。在莱顿载脂蛋白参考实验室,我们采用质谱策略详细了解蛋白质到肽的转化过程,以验证定量肽是否部分隐藏在错误裂解的蛋白质骨架中:方法:载脂蛋白(a)(apo(a))以非最佳方式消化,以增加通过高分辨率液相色谱串联质谱测量确定的误切肽的数量。仔细绘制了蛋白质到肽的转换图,并特别关注含有载脂蛋白(a)定量肽的误切肽。每个载脂蛋白(a)定量肽的四种不同同位素被用于评估特定实际样品测量过程中内部肽标准的线性度:结果:在基于质谱的apo(a)目标定量分析中,将隐藏在两种不同误解肽中的两种apo(a)定量肽纳入多反应监测列表,以提醒潜在的蛋白质消化差异。在应用我们的候选参考测量程序(RMP)进行载脂蛋白(a)定量时,可以排除误切肽的存在:这些数据进一步证实了我们的载脂蛋白(a)候选参考测量程序的有效性,它是国际临床化学与检验医学联合会认可的用于商业载脂蛋白(a)检测认证的高阶方法。基于质谱的分子检测和异质载脂蛋白(a)蛋白形式的定量将使制造商能够从混淆的脂蛋白(a)[Lp(a)]质量水平过渡到准确的摩尔载脂蛋白(a)水平。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Clinical chemistry and laboratory medicine
Clinical chemistry and laboratory medicine 医学-医学实验技术
CiteScore
11.30
自引率
16.20%
发文量
306
审稿时长
3 months
期刊介绍: Clinical Chemistry and Laboratory Medicine (CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. CCLM welcomes contributions on the progress in fundamental and applied research and cutting-edge clinical laboratory medicine. It is one of the leading journals in the field, with an impact factor over 3. CCLM is issued monthly, and it is published in print and electronically. CCLM is the official journal of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) and publishes regularly EFLM recommendations and news. CCLM is the official journal of the National Societies from Austria (ÖGLMKC); Belgium (RBSLM); Germany (DGKL); Hungary (MLDT); Ireland (ACBI); Italy (SIBioC); Portugal (SPML); and Slovenia (SZKK); and it is affiliated to AACB (Australia) and SFBC (France). Topics: - clinical biochemistry - clinical genomics and molecular biology - clinical haematology and coagulation - clinical immunology and autoimmunity - clinical microbiology - drug monitoring and analysis - evaluation of diagnostic biomarkers - disease-oriented topics (cardiovascular disease, cancer diagnostics, diabetes) - new reagents, instrumentation and technologies - new methodologies - reference materials and methods - reference values and decision limits - quality and safety in laboratory medicine - translational laboratory medicine - clinical metrology Follow @cclm_degruyter on Twitter!
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