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Digging for Missing Proteins Using Low-Molecular-Weight Protein Enrichment and a “Mirror Protease” Strategy
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2018-10-16 , DOI: 10.1021/acs.jproteome.8b00398 Cuitong He 1, 2 , Jinshuai Sun 2, 3 , Jiahui Shi 2, 3 , Yihao Wang 2 , Jialing Zhao 2, 4 , Shujia Wu 2, 4 , Lei Chang 2 , Huiying Gao 2 , Fengsong Liu 3 , Zhitang Lv 3 , Fuchu He 2 , Yao Zhang 1, 2 , Ping Xu 2, 3, 4
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2018-10-16 , DOI: 10.1021/acs.jproteome.8b00398 Cuitong He 1, 2 , Jinshuai Sun 2, 3 , Jiahui Shi 2, 3 , Yihao Wang 2 , Jialing Zhao 2, 4 , Shujia Wu 2, 4 , Lei Chang 2 , Huiying Gao 2 , Fengsong Liu 3 , Zhitang Lv 3 , Fuchu He 2 , Yao Zhang 1, 2 , Ping Xu 2, 3, 4
Affiliation
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In 2012, the Chromosome-centric Human Proteome Project (C-HPP) launched an investigation for missing proteins (MPs) to complete the Human Proteome Project (HPP). The majority of the MPs were distributed in low-molecular-weight (LMW) ranges, especially from 0 to 40 kDa. LMW protein identification is challenging, owing to their short length, low abundance, and hydrophobicity. Furthermore, many sequences from trypsin digestion are unlikely to yield detectable peptides or a reasonable quality of MS2 spectrum. Therefore, we focused on small MPs by combining LMW protein enrichment and a pair of complementary proteases strategy with trypsin and LysargiNase for human testis samples. In-depth testis LMW protein profiling resulted in the identification of 4063 proteins, of which 2565 were LMW proteins and 1130 had pairs of peptides generated from both trypsin and LysargiNase. This provided additional mass spectral evidence of further verification of small MPs. Finally, two MPs were verified from the seven MP candidates. One of them, Q8N688, was verified with two series of continuous and complementary b/y-product ions from the pairs of spectra for tryptic and LysargiNase digested peptides after the “mirror spectrum” matching. This make the confident identification of the representative peptides for the target MPs. On the contrary, the two verified peptides for Q86WR6 were identified with the same strategy from the gel-separation and gel-elution samples, respectively. Although the other five MP candidates showed high-quality spectra, they could not be sufficiently distinguished as PE1s and require further verification. All MS data sets have been deposited in the ProteomeXchange with identifier PXD010093.
中文翻译：
使用低分子量蛋白质富集和“镜像蛋白酶”策略挖掘缺失的蛋白质
2012年，以染色体为中心的人类蛋白质组计划（C-HPP）开展了一项针对缺失蛋白（MPs）的调查，以完成人类蛋白质组计划（HPP）。大多数MP分布在低分子量（LMW）范围内，尤其是0至40 kDa。由于LMW蛋白质的长度短，丰度低和具有疏水性，因此鉴定具有挑战性。此外，来自胰蛋白酶消化的许多序列不太可能产生可检测的肽或质量合理的MS 2光谱。因此，我们通过将LMW蛋白质富集和一对互补蛋白酶策略与胰蛋白酶和LysargiNase结合用于人睾丸，着眼于小型MP。深入睾丸LMW蛋白谱分析鉴定了4063种蛋白，其中2565种是LMW蛋白，而1130种具有成对的胰蛋白酶和LysargiNase肽。这为进一步验证小型MP提供了额外的质谱证据。最后，从七名国会议员候选人中选出了两名国会议员。其中一个Q8N688已通过“镜像光谱”匹配后，从胰蛋白酶和LysargiNase消化的肽光谱对中的两个连续且互补的b / y产物离子序列进行了验证。这样就可以确定目标MP的代表肽。相反，分别从凝胶分离和凝胶洗脱样品中以相同的策略鉴定了Q86WR6的两种验证肽。尽管其他五个MP候选者显示了高质量的光谱，但它们不能像PE1那样被充分区分，需要进一步验证。所有MS数据集均已存储在ProteomeXchange中，标识符为PXD010093。
更新日期：2018-10-17
中文翻译：
使用低分子量蛋白质富集和“镜像蛋白酶”策略挖掘缺失的蛋白质
2012年，以染色体为中心的人类蛋白质组计划（C-HPP）开展了一项针对缺失蛋白（MPs）的调查，以完成人类蛋白质组计划（HPP）。大多数MP分布在低分子量（LMW）范围内，尤其是0至40 kDa。由于LMW蛋白质的长度短，丰度低和具有疏水性，因此鉴定具有挑战性。此外，来自胰蛋白酶消化的许多序列不太可能产生可检测的肽或质量合理的MS 2光谱。因此，我们通过将LMW蛋白质富集和一对互补蛋白酶策略与胰蛋白酶和LysargiNase结合用于人睾丸，着眼于小型MP。深入睾丸LMW蛋白谱分析鉴定了4063种蛋白，其中2565种是LMW蛋白，而1130种具有成对的胰蛋白酶和LysargiNase肽。这为进一步验证小型MP提供了额外的质谱证据。最后，从七名国会议员候选人中选出了两名国会议员。其中一个Q8N688已通过“镜像光谱”匹配后，从胰蛋白酶和LysargiNase消化的肽光谱对中的两个连续且互补的b / y产物离子序列进行了验证。这样就可以确定目标MP的代表肽。相反，分别从凝胶分离和凝胶洗脱样品中以相同的策略鉴定了Q86WR6的两种验证肽。尽管其他五个MP候选者显示了高质量的光谱，但它们不能像PE1那样被充分区分，需要进一步验证。所有MS数据集均已存储在ProteomeXchange中，标识符为PXD010093。
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