Enhancement of Proteome Coverage by Ion Mobility Fractionation Coupled to PASEF on a TIMS-QTOF Instrument.
Enhancement of Proteome Coverage by Ion Mobility Fractionation Coupled to PASEF on a TIMS-QTOF Instrument.
复制标题
DOI：
10.1021/acs.jproteome.2c00336
复制
发表时间：
2022-08-05
影响因子：
4.4
通讯作者：
Stevens, Stanley M., Jr.
中科院分区：
文献类型：
--
作者：
Guergues, Jennifer;Wohlfahrt, Jessica;Stevens, Stanley M., Jr.
利用捕获离子迁移谱法（TIMS）在气相中根据离子迁移率（V·s/cm2）对离子进行分选，然后在四极杆飞行时间仪中进行平行积累-序列碎片化（PASEF），以确定对蛋白质组覆盖深度的影响。TIMS分离（多达4个气相部分）与DDA-PASEF结合，使用商业25 cm UPLC柱，90分钟梯度，每次注射200ng人（HeLa）细胞裂解液中检测到约7,000个蛋白质和超过70,000个肽。与默认的单一范围- tims DDA-PASEF分析相比，该结果对应于蛋白质和肽鉴定分别增加了约19%和30%。在技术重复的无标记定量中，平均CV值和中位数CV值均小于4%，表明TIMS分馏法不影响定量精度。利用TIMS分馏法生成基于dda的光谱库，用于使用较短的LC梯度对较低样本输入进行下游DIA分析。tims分离文库由超过7600个蛋白质和82000个肽组成，能够分别从10ng和200ng的人（HeLa）细胞裂解液输入中鉴定约4000和6600个蛋白质，梯度为20分钟，单次DIA分析。数据可在ProteomeXchange中获得：标识符PXD033129。
Trapped ion mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V·s/cm2) followed by parallel accumulation-serial fragmentation (PASEF) in a quadrupole time-of-flight instrument to determine the effect on the depth of proteome coverage. TIMS fractionation (up to 4 gas-phase fractions) coupled to DDA-PASEF resulted in the detection of ~7,000 proteins and over 70,000 peptides overall from 200ng of human (HeLa) cell lysate per injection using a commercial 25 cm UPLC column with a 90-minute gradient. This result corresponded to ~19 and 30% increases in protein and peptide identifications, respectively, when compared to a default, single range-TIMS DDA-PASEF analysis. Quantitation precision was not affected by TIMS fractionation as demonstrated by average and median CV values that were less than 4% upon label-free quantitation of technical replicates. TIMS fractionation was utilized to generate a DDA-based spectral library for downstream DIA analysis of lower sample input using a shorter LC gradient. The TIMS-fractionated library, consisting of over 7,600 proteins and 82,000 peptides, enabled the identification of ~4,000 and 6,600 proteins from 10ng and 200ng of human (HeLa) cell lysate input, respectively, with a 20-min gradient, single-shot DIA analysis. Data are available in ProteomeXchange: identifier PXD033129.
登录
查看更多内容
影响因子：
46.9
作者：
Washburn, MP;Wolters, D;Yates, JR
通讯作者：
Yates, JR
影响因子：
14.9
作者：
Perez-Riverol Y;Bai J;Bandla C;García-Seisdedos D;Hewapathirana S;Kamatchinathan S;Kundu DJ;Prakash A;Frericks-Zipper A;Eisenacher M;Walzer M;Wang S;Brazma A;Vizcaíno JA
通讯作者：
Vizcaíno JA
影响因子：
7
作者：
Bekker-Jensen, Dorte B.;Martinez-Val, Ana;Olsen, Jesper, V
通讯作者：
Olsen, Jesper, V
影响因子：
3.3
作者：
Guergues, Jennifer;Wohlfahrt, Jessica;Stevens, Stanley M., Jr.
通讯作者：
Stevens, Stanley M., Jr.
影响因子：
3.4
作者：
Guergues, Jennifer;Zhang, Ping;Stevens, Stanley M., Jr.
通讯作者：
Stevens, Stanley M., Jr.