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Instant on-paper protein digestion during blood spot sampling
Analyst ( IF 3.6 ) Pub Date : 2017-08-23 00:00:00 , DOI: 10.1039/c7an01075c Øystein Skjærvø 1, 2, 3, 4, 5 , Cecilie Rosting 1, 2, 3, 4, 5 , Trine Grønhaug Halvorsen 1, 2, 3, 4, 5 , Léon Reubsaet 1, 2, 3, 4, 5
Analyst ( IF 3.6 ) Pub Date : 2017-08-23 00:00:00 , DOI: 10.1039/c7an01075c Øystein Skjærvø 1, 2, 3, 4, 5 , Cecilie Rosting 1, 2, 3, 4, 5 , Trine Grønhaug Halvorsen 1, 2, 3, 4, 5 , Léon Reubsaet 1, 2, 3, 4, 5
Affiliation
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A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.
中文翻译：
血斑采样过程中即时的纸上蛋白质消化
本文介绍了结合采样和蛋白质消化的概念，结合了快速和简单的制造方法，即在蜡纸上用固定有胰蛋白酶的聚合物珠子在蜡纸上印刷蜡。纸质反应器显示出令人鼓舞的结果，可在五十分钟之内在模型蛋白质混合物以及人体血液中高度消化蛋白质。模型蛋白质混合物用于有或没有还原和烷基化步骤的性能评估。没有还原和烷基化的纸反应器显示了46％至75％的蛋白质序列覆盖率和5至20个高可信度肽段（分别为1和5个零缺失裂解肽段）。与传统的溶液方法相比，纸反应器的蛋白质序列覆盖率降低了10％，零缺失切割的高可信度肽减少29％，高可信度肽减少19％。蛋白质还原和烷基化步骤（在蛋白质消化之前或之后）的位置显示重要性不高。具有（六个星期）和不具有（十二个星期）胰蛋白酶肽的纸反应器的储存稳定性是令人满意的。纸质反应器消化复杂生物样品的能力通过与使用常规干血斑（DBS）程序制备的人类全血样品进行比较，并在非目标分析中进行了过夜消化，从而进行了比较。该反应器的蛋白质组性能为75±25，而DBS样品为76±5。此外，分别在纸上消解和DBS中鉴定出267±72和335±11个独特的肽（高可信度）。
更新日期：2017-09-12
中文翻译：
血斑采样过程中即时的纸上蛋白质消化
本文介绍了结合采样和蛋白质消化的概念，结合了快速和简单的制造方法，即在蜡纸上用固定有胰蛋白酶的聚合物珠子在蜡纸上印刷蜡。纸质反应器显示出令人鼓舞的结果，可在五十分钟之内在模型蛋白质混合物以及人体血液中高度消化蛋白质。模型蛋白质混合物用于有或没有还原和烷基化步骤的性能评估。没有还原和烷基化的纸反应器显示了46％至75％的蛋白质序列覆盖率和5至20个高可信度肽段（分别为1和5个零缺失裂解肽段）。与传统的溶液方法相比，纸反应器的蛋白质序列覆盖率降低了10％，零缺失切割的高可信度肽减少29％，高可信度肽减少19％。蛋白质还原和烷基化步骤（在蛋白质消化之前或之后）的位置显示重要性不高。具有（六个星期）和不具有（十二个星期）胰蛋白酶肽的纸反应器的储存稳定性是令人满意的。纸质反应器消化复杂生物样品的能力通过与使用常规干血斑（DBS）程序制备的人类全血样品进行比较，并在非目标分析中进行了过夜消化，从而进行了比较。该反应器的蛋白质组性能为75±25，而DBS样品为76±5。此外，分别在纸上消解和DBS中鉴定出267±72和335±11个独特的肽（高可信度）。
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