開發StageTip方法進行深度磷酸化蛋白質體分析

Abstract

蛋白質磷酸化是生物體內非常重要的蛋白質轉譯後修飾，影響眾多細胞訊息傳遞，以質譜技術為主的蛋白質體技術已經可對磷酸蛋白進行大規模鑑定與定量分析，然而目前樣品前處理的步驟繁瑣，易造成樣品損失及低再現性，難以運用在臨床樣品大規模分析處理。 為了能達到深度磷酸化蛋白質體分析與簡化處理步驟，我們致力於開發快速、可靠的樣品製備方法，利用StageTips (stop-and-go-extraction tips)開發一套完整磷酸蛋白質處理流程，包含從蛋白質提取、半胱胺酸殘基還原/烷基化反應、蛋白質酵素水解、除鹽、逆相層析分離胜肽到磷酸胜肽純化。 我們以人類非小細胞肺癌細胞PC9測試此磷酸蛋白質處理流程，開發的滴管尖內蛋白水解方法 (StageTip-based digestion)降低樣品處理過程時的流失，在質譜檢測上能比普遍使用的溶液內蛋白質水解方法(solution-based digestion) 鑑定到更多磷酸蛋白質，從1.5毫克PC9細胞蛋白，StageTip方法可測到8910條磷酸胜肽及2882個磷酸蛋白，而常用的溶液內水解方法僅得到7863條磷酸胜肽與2373個磷酸蛋白。 進一步評估方法的再現性，定量計算三重複實驗的所有磷酸胜肽的訊號強度，相較於溶液內水解方法(0.895-0.918)，StageTip 方法有良好的Pearson相關係數計算(0.924-0.945)，可證明有較高的樣品製備再現性。 我們挑選具有良好蛋白質萃取能力且能與蛋白酶相容的蛋白質變性試劑，並使用能快速反應的還原和烷基化試劑，使StageTip方法具有快速、簡單且高重複性的流程。為了更深度分析磷酸蛋白質體，我們將已建構好的蛋白質水解方法結合液相層析，將不同特性胜肽分群(fractionation)，進而降低樣品複雜性、提高質譜偵測磷酸蛋白質體之深度(磷酸蛋白質數量)。 StageTip內填裝的SDB-RPS同時具有反相與陽離子交換作用兩種親和力的性質，可將不同特性的胜肽分離。將胜肽分離為7個樣品群(fractions)再進行質譜分析，我們大幅增加偵測的磷酸胜肽(17962)以及磷酸蛋白(4204)，幾乎都是單次充提(9261磷酸胜肽，2828磷酸蛋白)的1.9及1.4倍以上的數量，並有較寬的偵測動態範圍(dynamic range)，能偵測到更低含量的胜肽。利用最新一代質譜儀LTQ Orbitrap Fusion Lumos，在1%錯誤發現率的條件下，相同樣品更可鑑定到高達28022條磷酸胜肽與5338個磷酸蛋白。 接著，我們進一步串聯蛋白質酵素水解、胜肽分離及固定金屬離子親合性層析做磷酸胜肽純化之兩種StageTip，透過調整兩系統相容的溶液種類與酸鹼值，離心分離沖提下來的胜肽可以直接進入固定金屬離子親合性層析的StageTip內，進行磷酸胜肽的純化。透過此方法，從1.5 毫克的細胞蛋白可偵測到25,311條磷酸胜肽與5073個磷酸蛋白，此外利用4.5毫克的細胞蛋白更可得到30,329個蛋白磷酸化位置，與目前報導最大規模磷酸蛋白分析的文獻相比(38,229個蛋白磷酸化位置)，他們總共利用14天的質譜時間分析，而我們的方法僅需4.5天，顯示串聯StageTip為高靈敏度具有深度磷酸蛋白的分析方法。

Protein phosphorylation is one of the most essential post-translation modifications (PTMs) regulating cellular signaling processes. Shotgun proteomics has become an indispensable tool to allow identification and quantitation of thousands of phosphosites in variety of sample. However, the multi-step sample preparation required for phosphoproteomics is labor-intensive and prone to sample loss and poor reproducibility, posing a challenge for large-scale analysis of clinical samples with limited amount of material. Toward deep and simplified shotgun phosphoproteomic profiling, we implemented the stop-and-go-extraction tips (StageTips) to develop a streamlined phosphoproteomic workflow from protein extraction, protein reduction/alkylation, proteolytic digestion, peptide clean up, peptide fractionation, followed by StageTip-based IMAC for enrichment of phosphopeptides. We firstly optimize the compatibility between solubilization reagent and proteases, sodium laurate is a strong but protease compatible detergent which was selected and incorporated into rapid reducing/alkylating reagent (TCEP/CAA). The use of sodium laurate and TCEP/CAA had good performance of phosphoproteome identification and rapid, simple workflow compared to common method. The feasibility of this protocol was demonstrated on non-small cell lung cancer cell lines, PC9. On the analysis of 1.5 mg PC9 cell lysate, StageTip-based digestion showed slightly superior performance (8910 phosphopeptides from 2882 phosphoproteins) compared to conventional in-solution digestion (7863 phosphopeptides from 2373 phosphoproteins) from triplicate analysis. The reproducibility of this protocol was evaluated by quantitative comparison of phosphopeptide abundance obtained by extracted ion chromatogram in LC-MS/MS; StageTip-based digestion showed superior Pearson correlation coefficient (0.924-0.945) compared to the solution-based digestion (MeOH/CHCl3 precipitation) (0.895-0.918). Toward in-depth phosphoproteomic analysis, we also incorporated peptide fractionation strategy into the StageTip and each fraction was subjected to phosphopeptide enrichment by IMAC. The copolymeric resin particle SDB-RPS (sulfonated styrene-divinylbenzene reversed phase), which exhibits features of both reversed phase and cation exchange interactions, was packed in StageTip. The phosphopeptides fractionation was performed into 7 fractions. Compared to the identification result of 9,261 phosphopeptide (2,828 phosphoproteins) from single elution, 1.9-fold and 1.4-fold more phosphopeptides and phosphoproteins were observed from the StageTip fractionation strategy (17,962 phosphopeptide from 4,204 phosphoproteins). With the latest generation LTQ Orbitrap Fusion Lumos mass spectrometer, 28,022 phosphopeptides from 5,338 phosphoproteins were identified at 1% FDR (false discovery rate). Furthermore, we assembled two main steps, StageTip digestion and StageTip-based IMAC enrichment. By optimizing the buffer type and pH condition for the two StageTip systems, the fractionated peptides in each fraction could directly elute into IMAC StageTip using co-centrifugation, which bypassed the drying step to reduce sample loss. A total 25,311 phosphopeptides from 5,073 phosphoproteins were identified (1% FDR) from 1.5 mg cell lysate. Using total of 4.5 mg cell lysate for triplicate LC-MS/MS runs, 30,329 phosphosites were obtained (1% FDR). The result was almost comparable to the deepest phosphoproteome analysis to date (38,229 phosphosites). Our approach greatly reduced the analysis time (4.5 days) compared to their long LC-MS/MS time (14 days). Taken together, this streamline protocol is capable of achieving deep phosphoproteomic analysis with high sensitivity from limited samples.