Dufresne Jaimie, Chen Zhuo Zhen, Sehajpal Pallvi, Bowden Peter, Ho Ja-An, Hsu Cheng-Chih Richard, Marshall John G
Department of Chemistry and Biology, Faculty of Science, Toronto Metropolitan University, 350 Victoria Street, Toronto, Ontario M5B 2K3, Canada.
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
ACS Omega. 2025 Jan 2;10(1):281-293. doi: 10.1021/acsomega.4c05624. eCollection 2025 Jan 14.
Naturally occurring peptides display a wide mass distribution after ionization due to the presence of heavy isotopes of C, H, N, O, and S and hydrogen loss. There is a crucial need for sensitive methods that collect as much information as possible about all plasma peptide forms. Statistical analysis of the delta mass distribution of peptide precursors from MS/MS spectra that were matched to 63,077 peptide sequences by X!TANDEM revealed Gaussian peaks representing heavy isotopes and hydrogen loss at integer delta mass values of -3, -2, -1, 0, +1, +2, +3, +4, and +5 Da. Human plasma samples were precipitated in acetonitrile, and the resulting proteins were collected over a quaternary amine resin, eluted with NaCl, digested with trypsin, and analyzed by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with an orbital ion trap (OIT). Fragment spectra (MS/MS) generated from the OIT data were fit to human fully tryptic peptides by X!TANDEM, which led to the identification of 3,888 protein gene symbols represented by three or more peptides ( ≥ 3). The peptide counts to plasma proteins from experimental MS/MS spectra were corrected against 29 blank LC-ESI-MS/MS spectra and 30 million random MS/MS control spectra to yield 2,784 true positive proteins ( ≥ 3; ≤ 0.01). Peptides identified by fragmenting ions with Gaussian heavy isotopes and hydrogen loss that were matched to known plasma proteins, such as albumin (ALB), were shown to be true positives and agreed with the peptide sequences identified in the monoisotopic peak. Accepting the ions from the monoisotopic peak alone (±0.1 Da) yielded only 382 plasma proteins ( ≥ 3; type I error ≤ 0.01; type II error ∼86%). In contrast, accepting all ions within ±0.1 Da around the hydrogen loss, monoisotopic, and heavy isotopic peaks led to the identification of 963 proteins ( ≥ 3; ≤ 0.01; type II error ∼60%). Using the power of the OIT to resolve the Gaussian peaks from heavy isotopes and hydrogen loss resulted in the identification of three times more proteins with high confidence and a much lower type II error than analyzing peptides from the monoisotopic peak alone. The resolving power of the OIT may be exploited to increase observation frequencies and provide greater proteomic coverage and statistical power in comparative proteomics studies.
由于存在碳、氢、氮、氧和硫的重同位素以及氢损失，天然存在的肽在电离后显示出广泛的质量分布。迫切需要灵敏的方法来收集尽可能多的关于所有血浆肽形式的信息。通过X!TANDEM与63,077个肽序列匹配的MS/MS谱图中肽前体的δ质量分布的统计分析显示，在整数δ质量值-3、-2、-1、0、+1、+2、+3、+4和+5 Da处有代表重同位素和氢损失的高斯峰。人血浆样品用乙腈沉淀，所得蛋白质收集在季胺树脂上，用氯化钠洗脱，用胰蛋白酶消化，并用带轨道离子阱（OIT）的纳升液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）进行分析。由OIT数据生成的碎片谱（MS/MS）通过X!TANDEM与人类完全胰蛋白酶消化的肽进行拟合，从而鉴定出由三种或更多种肽（≥3）代表的3,888个蛋白质基因符号。将实验MS/MS谱图中血浆蛋白的肽计数与29个空白LC-ESI-MS/MS谱图和3000万个随机MS/MS对照谱图进行校正，得到2784个真阳性蛋白（≥3；≤0.01）。通过对与已知血浆蛋白（如白蛋白（ALB））匹配的具有高斯重同位素和氢损失的离子进行碎片化鉴定的肽被证明是真阳性，并且与在单同位素峰中鉴定的肽序列一致。仅接受来自单同位素峰（±0.1 Da）的离子仅产生382种血浆蛋白（≥3；I型错误≤0.01；II型错误约86%）。相比之下，接受氢损失、单同位素和重同位素峰周围±0.1 Da范围内的所有离子导致鉴定出963种蛋白质（≥3；≤0.01；II型错误约60%）。利用OIT分辨重同位素和氢损失产生的高斯峰的能力，与仅分析单同位素峰的肽相比，能够以高置信度鉴定出多三倍的蛋白质，并且II型错误要低得多。OIT的分辨能力可用于提高观察频率，并在比较蛋白质组学研究中提供更大的蛋白质组覆盖范围和统计能力。
