Automated Precursor Ion Exclusion During LC-MS/MS Data Acquisition for Optimal Ion Identification

Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for characterizing multiple samples of complex mixtures with similar compositions. This article addresses a data acquisition strategy for collecting a maximal number of unique, high-quality MS/MS during LC-MS/MS analysis of multiple samples. Based on the concept that a component only needs to be identified once when analyzing multiple samples with similar compositions, an automated intersample data-dependent acquisition strategy was developed. The strategy is based on precursor ion exclusion (PIE) and is implemented in MassAnalyzer in an automated fashion for Thermo Scientific (San Jose, CA, USA) mass spectrometers. In this method, MassAnalyzer submits one sample at a time to the sample queue. After data acquisition of each sample, MassAnalyzer automatically analyzes the data to generate a PIE list based on the MS/MS precursor ions, merges this list with the list generated from previous runs, adds the list to the MS method file, and submits the next sample to the queue. The PIE list contains both m/z value and time window for each precursor ion, and is generated intelligently so that if an MS/MS is insufficient for identifying the peak of interest, it will be collected again near the top of the peak in the next run. Therefore, the strategy maximizes both quality and the number of unique MS/MS. When automated PIE was used to acquire LC-MS/MS data of an antibody tryptic digest and a soy hydrolysate sample, the number of identified ions increased by 52 % and 93 %, respectively, compared with data acquired without using PIE.

Impact of data‐dependent exclusion list based mass spectrometry on label‐free proteomic quantification

“…The 'dynamic exclusion' principle is employed during DDA to identify the maximum number of ions which temporarily puts the m/z value of an ion into an exclusion list for a set length of time after its MS/MS spectrum has been acquired once. As a result, the instrument will spend more time acquiring MS/MS spectra of less abundant ions instead of continuously acquiring the same ion's MS/MS spectrum . Again, all co‐eluting peptides at a given retention time are not subjected to MS/MS due to the limited spectral acquisition speed of the mass spectrometer.…”

Metabolomics analysis of soy hydrolysates for the identification of productivity markers of mammalian cells for manufacturing therapeutic proteins

“…These precursor rejection lists were generated by MassAnalyzer and applied to later samples. This inter‐sample data‐dependent acquisition strategy has been shown to increase the number of identified compounds significantly …”

“…Many of the algorithms utilized in this work have been described previously, including feature extraction/charge determination, retention time alignment, and de novo sequencing . Feature extraction was performed on all ions with signal‐to‐noise ratio above 4, with noise level estimated from the distribution of all signals in a run as described previously …”

MZDASoft: a software architecture that enables large‐scale comparison of protein expression levels over multiple samples based on liquid chromatography/tandem mass spectrometry

“…In addition, when the multiplexing capacity is reached, protein comparison coverage will reach its limit. Solutions based on spectral counting in label‐free tandem MS suffer from large quantification variations . Recently, data‐independent acquisition (DIA) has received much interest, which fragments all peptides and generates reproducible peptide fragment maps.…”

“…Solutions based on spectral counting in label-free tandem MS [7,8] suffer from large quantification variations. [7,9,10] Recently, dataindependent acquisition (DIA) [11][12][13][14] has received much interest, which fragments all peptides and generates reproducible peptide fragment maps. Although the total number of peptide identifications of DIA is similar to that of tandem MS per run, [15] the protein comparison coverage over several runs can increase significantly.…”