Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 E Fowler Avenue, Tampa, Florida 33620, United States.
J Proteome Res. 2022 Aug 5;21(8):2036-2044. doi: 10.1021/acs.jproteome.2c00336. Epub 2022 Jul 24.
Trapped ion-mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V s/cm), followed by parallel accumulation-serial fragmentation (PASEF) using a quadrupole time-of-flight instrument to determine the effect on the depth of proteome coverage. TIMS fractionation (up to four gas-phase fractions) coupled to data-dependent acquisition (DDA)-PASEF resulted in the detection of ∼7000 proteins and over 70,000 peptides overall from 200 ng of human (HeLa) cell lysate per injection using a commercial 25 cm ultra high performance liquid chromatography (UHPLC) column with a 90 min 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 the average and median coefficient of variation 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 data-independent acquisition (DIA) analysis of lower sample input using a shorter LC gradient. The TIMS-fractionated library, consisting of over 7600 proteins and 82,000 peptides, enabled the identification of ∼4000 and 6600 proteins from 10 and 200 ng of human (HeLa) cell lysate input, respectively, with a 20 min gradient, single-shot DIA analysis. Data are available in ProteomeXchange: identifier PXD033129.
采用离子淌度谱（TIMS）对气相中的离子根据其离子淌度（V s/cm）进行分离，然后使用四极杆飞行时间仪器进行平行累积-串联碎裂（PASEF），以确定对蛋白质组覆盖深度的影响。TIMS 分级（最多四级气相级分）与数据依赖采集（DDA）-PASEF 相结合，从每次注射 200ng 人（HeLa）细胞裂解物中总共检测到约 7000 种蛋白质和超过 70000 种肽，使用商业 25cm 超高效液相色谱（UHPLC）柱进行 90min 梯度洗脱。与默认的单范围 TIMS DDA-PASEF 分析相比，这分别对应于蛋白质和肽鉴定的约 19%和 30%的增加。TIMS 分级不会影响定量精度，这可以通过无标签定量技术重复的平均和中位数变异系数值小于 4%来证明。TIMS 分级用于生成基于 DDA 的光谱库，以便在使用较短的 LC 梯度对较低样本输入进行下游数据独立采集（DIA）分析时使用。TIMS 分级的文库由超过 7600 种蛋白质和 82000 种肽组成，能够从每次注射 10ng 和 200ng 人（HeLa）细胞裂解物中鉴定出约 4000 种和 6600 种蛋白质，梯度为 20min，单喷 DIA 分析。数据可在 ProteomeXchange 中获得：标识符 PXD033129。
