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用无标记蛋白质组学方法定量分析家鼠中高度同源的主要尿蛋白的调控。

Regulation of highly homologous major urinary proteins in house mice quantified with label-free proteomic methods.

作者信息

Enk Viktoria M, Baumann Christian, Thoß Michaela, Luzynski Kenneth C, Razzazi-Fazeli Ebrahim, Penn Dustin J

机构信息

VetCore-Facility for Research, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210-Vienna, Austria.

出版信息

Mol Biosyst. 2016 Oct 20;12(10):3005-16. doi: 10.1039/c6mb00278a. Epub 2016 Jul 28.

DOI:10.1039/c6mb00278a
PMID:27464909
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5166567/
Abstract

Major urinary proteins (MUPs) are highly homologous proteoforms that function in binding, transporting and releasing pheromones in house mice. The main analytical challenge for studying variation in MUPs, even for state-of-the-art proteomics techniques, is their high degree of amino acid sequence homology. In this study we used unique peptides for proteoform-specific identification. We applied different search engines (ProteinPilot™vs. PEAKS®) and protein databases (MUP database vs. SwissProt + unreviewed MUPs), and found that proteoform identification is influenced by addressing background proteins (unregulated urinary proteins, non-MUPs) during the database search. High resolution Q-TOF mass spectrometry was used to identify and precisely quantify the regulation of MUP proteoforms in male mice that were reared in standard housing and then transferred to semi-natural enclosures (within-subject design). By using a designated MUP database we were able to distinguish 19 MUP proteoforms, with A2CEK6 (a Mup11 gene product) being the most abundant based on spectral intensities. We compared three different quantification strategies based on MS1- (from IDA and SWATH™ spectra) and MS2 (SWATH™) data, and the results of these methods were correlated. Furthermore, three data normalization methods were compared and we found that increased statistical significance of fold-changes can be achieved by normalization based on urinary protein concentrations. We show that male mice living in semi-natural enclosures significantly up-regulated some but not all MUPs (differential regulation), e.g., A2ANT6, a Mup6 gene product, was upregulated between 9-fold (MS1) and 13-fold (MS2) using the designated MUP database. Finally, we show that 85 ± 7% of total MS intensity can be attributed to MUP-derived peptides, which supports the assumption that MUPs are the primary proteins in mouse urine. Our results provide new tools for assessing qualitative and quantitative variation of MUPs and suggest that male mice regulate the expression of specific MUP proteoforms, depending upon social conditions.

摘要

主要尿蛋白(MUPs)是高度同源的蛋白质异构体,在小家鼠中发挥结合、运输和释放信息素的功能。即使对于最先进的蛋白质组学技术而言,研究MUPs变异的主要分析挑战在于它们高度的氨基酸序列同源性。在本研究中,我们使用独特肽段进行蛋白质异构体特异性鉴定。我们应用了不同的搜索引擎(ProteinPilot™与PEAKS®)和蛋白质数据库(MUP数据库与SwissProt + 未审核的MUPs),发现蛋白质异构体鉴定受数据库搜索过程中处理背景蛋白(未调控的尿蛋白、非MUPs)的影响。高分辨率Q-TOF质谱用于鉴定并精确量化在标准饲养环境中饲养后转移至半自然围栏中的雄性小鼠体内MUP蛋白质异构体的调控情况(受试者内设计)。通过使用指定的MUP数据库,我们能够区分出19种MUP蛋白质异构体,基于光谱强度,A2CEK6(一种Mup11基因产物)最为丰富。我们比较了基于MS1(来自IDA和SWATH™光谱)和MS2(SWATH™)数据的三种不同定量策略,这些方法的结果具有相关性。此外,比较了三种数据归一化方法,我们发现基于尿蛋白浓度进行归一化可提高倍数变化的统计显著性。我们发现生活在半自然围栏中的雄性小鼠显著上调了部分而非全部MUPs(差异调控),例如,使用指定的MUP数据库时,Mup6基因产物A2ANT6上调了9倍(MS1)至13倍(MS2)。最后,我们表明总MS强度的85±7%可归因于源自MUP的肽段,这支持了MUPs是小鼠尿液中主要蛋白质的假设。我们的结果为评估MUPs的定性和定量变异提供了新工具,并表明雄性小鼠根据社会条件调节特定MUP蛋白质异构体的表达。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/5b71d207b838/c6mb00278a-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/a7abb549655d/c6mb00278a-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/fdbcbaa50011/c6mb00278a-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/4fca8ae06b07/c6mb00278a-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/290411bb11b8/c6mb00278a-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/5b71d207b838/c6mb00278a-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/a7abb549655d/c6mb00278a-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/fdbcbaa50011/c6mb00278a-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/4fca8ae06b07/c6mb00278a-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/290411bb11b8/c6mb00278a-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c3a7/5166567/5b71d207b838/c6mb00278a-f5.jpg

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Methods for SWATH™: Data Independent Acquisition on TripleTOF Mass Spectrometers.
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Pheromones that correlate with reproductive success in competitive conditions.与竞争条件下生殖成功相关的信息素。
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Biological Roles of Lipocalins in Chemical Communication, Reproduction, and Regulation of Microbiota.脂质运载蛋白在化学通讯、生殖及微生物群调节中的生物学作用
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