iTRAQ Proteomic Analysis of Wheat (Triticum aestivum L.) Genotypes Differing in Waterlogging Tolerance

Strong cation exchange (SCX) fractionation and LC-MS/MS analysis
The combined labeled samples were bound to a SCX fractionation column connected with a high performance liquid chromatography (HPLC) system. The peptide mixture was re-dissolved in the buffer A (20 mM ammonium formate in water, pH10.0), and then fractionated by high pH separation using Ultimate 3000 system (Thermo Fisher scientific, MA, USA) connected to a reverse phase column (Gemini-NX 3u C18 110A column, 2.0 mm x 150 mm, 3 μm, (Waters Corporation, MA, USA). High pH separation was performed using a linear gradient starting from 5% to 45% buffer B (20 mM ammonium formate in 80% ACN, pH 10.0) in 40 min. The column flow rate was maintained at 0.2 mL/min and column temperature was maintained at 30℃. A total of 12 fractions were collected, and each fraction was dried in a vacuum concentrator for the next step.
Peptide fractions were resuspended with 30 μL solvent C ( water with 0.1% formic acid), respectively, and separated by nanoLC and analyzed by electrospray tandem mass spectrometry. The experiments were performed on an Easy-nLC 1000 system (Thermo Fisher Scientific, MA, USA). A total of 10 μL peptide sample was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 μm × 2 cm), with a flow of 10 μL/min for 3 min and subsequently separated on the analytical column (Acclaim PepMap C18, 75 μm × 15 cm) with a linear gradient, from 3% to 32% solvent D (ACN with 0.1% formic acid) in 120 min. The column flow rate was maintained at 300 nL/min.
The fusion mass spectrometer was run in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 350-1550) were acquired with a mass resolution of 120 K, followed by sequential high energy collisional dissociation MS/MS scans with a resolution of 30 K. The isolation window was set as 1.6 Da. MS/MS fixed first mass was set at 110. In all cases, one microscan was recorded using dynamic exclusion of 45 seconds.
Database search and Quantification
The mass spectrometry data were transformed into MGF (Mascot generic format) files with Proteome Discovery 1.2 (Thermo, PA, USA) and analyzed using Mascot software version 2.3.2 (Matrix Science, London, UK). Mascot database was set up for protein identification using Triticum aestivum L database in NCBI nr (release 2017_03); SwissProt/UniprotKB (release 2018_06) and International Protein Index (IPI; version 3.16). Trypsin/P was chosen as the enzyme with two missed cleavages allowed; Peptide tolerance was set at 10 ppm, and Mascot was searched with a fragment ion mass tolerance of 0.050 Da; a parent ion tolerance of 10.0 PPM. Significance threshold p < 0.05 (with 95% confidence). The average values of the biological replicates were used to indicate the final protein abundances for each sample. Proteins with a 1.2-fold change between samples and a p value less than 0.05 were determined as differentially expressed proteins (DEPs).

强阳离子交换（Strong cation exchange, SCX）分级分离与液相色谱-串联质谱（LC-MS/MS）分析
标记后的混合样品结合至与高效液相色谱（high performance liquid chromatography, HPLC）系统相连的SCX分级色谱柱。将肽混合物复溶于缓冲液A（20 mM甲酸铵水溶液，pH 10.0），随后通过连接有反相色谱柱（Gemini-NX 3u C18 110A柱，2.0 mm × 150 mm，3 μm，沃特世公司，美国马萨诸塞州）的Ultimate 3000系统（赛默飞世尔科技，美国马萨诸塞州）进行高pH梯度分级分离。高pH分级采用线性洗脱梯度，在40 min内从5%缓冲液B（20 mM甲酸铵溶于80%乙腈，pH 10.0）升至45%缓冲液B。色谱柱流速维持在0.2 mL/min，柱温恒定为30℃。共收集12个分级组分，每个组分经真空浓缩仪干燥后用于后续实验。
肽段分级组分分别以30 μL溶剂C（含0.1%甲酸的水溶液）重悬，经纳升液相色谱（nanoLC）分离后通过电喷雾串联质谱进行分析。实验采用Easy-nLC 1000系统（赛默飞世尔科技，美国马萨诸塞州）完成。取10 μL肽段样品上样至捕集柱（赛默飞世尔Acclaim PepMap C18，100 μm × 2 cm），以10 μL/min的流速洗脱3 min，随后在分析柱（Acclaim PepMap C18，75 μm × 15 cm）上以线性梯度分离：120 min内从3%溶剂D（含0.1%甲酸的乙腈溶液）升至32%溶剂D。色谱柱流速维持在300 nL/min。
该联用质谱仪以数据依赖模式运行，可自动在MS和MS/MS采集模式间切换。一级全扫描质谱（m/z 350-1550）的质量分辨率设为120 K，随后依次进行高能碰撞解离MS/MS扫描，分辨率设为30 K。隔离窗口设置为1.6 Da，MS/MS的固定第一质量设为110。所有采集均采用单次微扫，动态排除时间设为45秒。
数据库搜索与定量分析
利用Proteome Discovery 1.2（赛默飞，美国宾夕法尼亚州）将质谱原始数据转换为Mascot通用格式（Mascot generic format, MGF）文件，采用Mascot软件v2.3.2（Matrix Science，英国伦敦）进行数据分析。以NCBI nr数据库（2017_03版）、SwissProt/UniprotKB数据库（2018_06版）以及国际蛋白质索引（IPI；v3.16）中的普通小麦（Triticum aestivum L）数据库作为检索库完成蛋白质鉴定。酶解规则选择胰蛋白酶/P（Trypsin/P），允许2个漏切位点；肽段质量容忍度设为10 ppm，碎片离子质量容忍度设为0.050 Da，母离子质量容忍度为10.0 ppm。显著性阈值设定为p < 0.05（置信度95%）。以生物学重复的平均值表征每个样品的最终蛋白质丰度。将样品间表达量变化倍数达1.2倍且p值小于0.05的蛋白质鉴定为差异表达蛋白质（differentially expressed proteins, DEPs）。