Agrawal Neha, Govekar Rukmini
Advanced Centre for Treatment, Research, and Education in Cancer (ACTREC) Navi Mumbai India.
Rukmini Lab Homi Bhabha National Institute Mumbai India.
Anal Sci Adv. 2024 Dec 20;6(1):e202400058. doi: 10.1002/ansa.202400058. eCollection 2025 Jun.
Cell signalling is a vital process in cell physiology, which is driven by protein phosphorylation. Global phosphoproteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has thus gained importance in cell signalling research. However, phosphoprotein identification by LC-MS/MS in whole cell lysates, which are complex protein mixtures, is hindered by their poor ionization coupled with suppression of peaks due to low abundance. Enrichment by immobilized metal ion- and metal oxide-affinity chromatography (IMAC and MOAC), which preferentially enrich multi- and mono-phosphorylated proteins, respectively, have improved their detection by MS. However, preferential enrichment limits phosphoproteome coverage in global analyses of cell lysates which contain mono- and multi-phosphorylated proteins. Improvement in their coverage by sequential elution approach that exploits the complementary chemistries of these matrices has been reported. In this study, we observed that the number of phosphoproteins detected using the sequential elution approach was lower (∼250-400) as compared to the theoretically predicted number (>500) based on their reported 30% abundance in the cell proteome (1700-2200 proteins detected by MS in our cell lines). Acknowledging the merit of using multiple matrices, we used IMAC and MOAC individually and pooled the data. We observed a remarkable increase (>30%) in phosphoproteome coverage. Further, though 98% of phosphoproteins were enriched by IMAC, among the remaining 2%, those detected exclusively by MOAC were biologically important. This justified the use of multiple matrices. Thus, an incremental modification of using multiple matrices individually rather than sequentially and pooling the data markedly improved the phosphoproteome coverage, which can positively impact cell signalling research.
细胞信号传导是细胞生理学中的一个重要过程，由蛋白质磷酸化驱动。因此，通过液相色谱 - 串联质谱法（LC-MS/MS）进行的全球磷酸化蛋白质组分析在细胞信号传导研究中变得越来越重要。然而，在全细胞裂解物（复杂的蛋白质混合物）中通过LC-MS/MS鉴定磷酸化蛋白质受到其电离性差以及由于丰度低导致的峰抑制的阻碍。通过固定化金属离子亲和色谱法（IMAC）和金属氧化物亲和色谱法（MOAC）进行富集，分别优先富集多磷酸化和单磷酸化蛋白质，提高了它们通过质谱检测的能力。然而，在含有单磷酸化和多磷酸化蛋白质的细胞裂解物的全局分析中，优先富集限制了磷酸化蛋白质组的覆盖范围。据报道，通过利用这些基质的互补化学性质的顺序洗脱方法可以提高其覆盖范围。在本研究中，我们观察到，与基于其在细胞蛋白质组中报道的30%丰度理论预测的数量（>500）相比，使用顺序洗脱方法检测到的磷酸化蛋白质数量较低（约250 - 400）（我们的细胞系中通过质谱检测到1700 - 2200种蛋白质）。认识到使用多种基质的优点，我们分别使用IMAC和MOAC并汇总数据。我们观察到磷酸化蛋白质组覆盖范围显著增加（>30%）。此外，虽然98%的磷酸化蛋白质通过IMAC富集，但在其余2%中，那些仅由MOAC检测到的蛋白质具有生物学重要性。这证明了使用多种基质的合理性。因此，单独而非顺序使用多种基质并汇总数据的渐进性改进显著提高了磷酸化蛋白质组的覆盖范围，这可以对细胞信号传导研究产生积极影响。
