Drosophila melanogaster iTRAQ HCD dataset

Quantitative comparation of the tagmata of body and head using iTRAQ based on HCD Fragmentation. The 8-plex iTRAQ Multiplex Buffer Kit (AB SCIEX, Foster City, CA) was used to label different peptide fractions, in which iTRAQ reagents dissolved in isopropyl alcohol. Another internal standard of D.melanogaster body and head mixed with 1:1 (B+H) was also used when doing iTRAQ labeling. Therefore the same amount of B (body), H1 (head), H2 (head), B+H (internal standard) peptide fractions prepared as described above were labeled by equal but different iTRAQ reagents and incubated for 5 h at room temperature. The four different labeled peptide fractions (B:113, H1:114, H2:115, B+H:116 ) were then mixed with 1:1 and dried in a speedvac followed by desalting purification using stage tip. All prepared peptides were further analyzed on an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Electron, San Jose, CA) coupled with UPLC (nano Acquity Ultra Performance LC, Waters). For HCD raw files, the profile data was firstly centralized by ReAdW.exe in TPP and then deisotoped and deconvoluted using in-house made scripts to improve the identification rate of spectra. All MGF files were searched using Mascot 2.3 against a Drosophila melanogaster database with 24,043 entries (http://flybase.org/, release 5.4, 24,043 entries). The target-decoy based strategy was used to control the peptide false discovery rate (FDR).基于高能碰撞诱导解离（HCD）技术，采用同位素标记相对和绝对定量（iTRAQ）对黑腹果蝇（Drosophila melanogaster）躯体与头部体区开展定量比较分析。本研究使用8标级iTRAQ多重缓冲液试剂盒（AB SCIEX，美国加利福尼亚州福斯特城）对不同肽段组分进行标记，该试剂盒中的iTRAQ试剂均溶解于异丙醇中。在进行iTRAQ标记时，同步引入按1:1比例混合的黑腹果蝇躯体与头部样品作为内标（B+H）。据此，按前述方法制备的等质量躯体（B）、头部重复样本1（H1）、头部重复样本2（H2）及内标混合样（B+H）的肽段组分，分别使用等量但不同标签的iTRAQ试剂标记，并于室温孵育5小时。将上述四种经不同iTRAQ试剂标记的肽段组分（B:113、H1:114、H2:115、B+H:116）按1:1比例混合后，经真空离心浓缩仪干燥，再采用StageTip固相萃取微柱进行脱盐纯化。将所有制备完成的肽段，通过与超高效液相色谱（UPLC，Waters nano Acquity超高性能液相色谱系统）联用的LTQ-Orbitrap Velos混合型质谱仪（赛默飞世尔电子（Thermo Electron），美国加利福尼亚州圣何塞市）进行后续分析。针对HCD原始数据文件，首先使用转录蛋白质组学流程（Trans-Proteomic Pipeline，TPP）中的ReAdW.exe程序对原始轮廓数据进行中心化处理，随后采用自研脚本完成去同位素峰与解卷积操作，以提升质谱谱图的鉴定率。所有质谱通用格式（MGF）文件均通过Mascot 2.3搜索引擎，针对包含24043条蛋白序列的黑腹果蝇数据库（FlyBase，版本5.4，共24043条条目，网址：http://flybase.org/）进行检索。本研究采用靶标-诱饵数据库策略，对肽段鉴定的假阳性发现率（FDR）进行质控。