Optimization of Acquisition and Data-Processing Parameters for Improved Proteomic Quantification by Sequential Window Acquisition of All Theoretical Fragment Ion Mass Spectrometry

Proteomic analysis with data-independent acquisition (DIA) approaches represented by the sequential window acquisition of all theoretical fragment ion spectra (SWATH) technique has gained intense interest in recent years because DIA is able to overcome the intrinsic weakness of conventional data-dependent acquisition (DDA) methods and afford higher throughout and reproducibility for proteome-wide quantification. Although the raw mass spectrometry (MS) data quality and the data-mining workflow conceivably influence the throughput, accuracy and consistency of SWATH-based proteomic quantification, there lacks a systematic evaluation and optimization of the acquisition and data-processing parameters for SWATH MS analysis. Herein, we evaluated the impact of major acquisition parameters such as the precursor mass range, isolation window width and accumulation time as well as the data-processing variables including peak extraction criteria and spectra library selection on SWATH performance. Fine tuning these interdependent parameters can further improve the throughput and accuracy of SWATH quantification compared to the original setting adopted in most SWATH proteomic studies. Furthermore, we compared the effectiveness of two widely used peak extraction software PeakView and Spectronaut in discovery of differentially expressed proteins in a biological context. Our work is believed to contribute to a deeper understanding of the critical factors in SWATH MS experiments and help researchers optimize their SWATH parameters and workflows depending on the sample type, available instrument and software

Additional file 1 of Network meta-analysis on efficacy and safety of different biologics for ulcerative colitis

Additional file 1

Primers used for cloning and qPCR in this study.

<p>Primers used for cloning and qPCR in this study.</p

qRT-PCR analysis of CcTrx1 tissue-specific expression.

<p>Relative expression was calculated using the 2^−ΔΔCt method with GAPDH as the reference gene. The results are presented as the relative quantity values. All treatments were performed in triplicate, and data were presented as mean ± SE (n = 3, ** <i>P</i><0.01 <i>vs</i>. tentacle).</p

Expression and purification of the rCcTrx1 fusion protein.

<p>(A) 12% SDS-PAGE analysis of the samples collected from different steps of expression and purification. Lane 1, whole cell lysates of recombinant <i>E. coli</i> BL21 (DE3) before induction; lane 2, whole cell lysates of recombinant <i>E. Coli</i> BL21 (DE3) after induction with 1 mM IPTG for 8 h at 25°C; lane 3, fractions from the 30 mM imidazole wash of the HisTrap HP affinity column; lane 4, fractions from the 500 mM imidazole elution of the HisTrap HP affinity column. The position corresponding to the rCCTrx1 protein is indicated by an arrow. (B) Western blotting analysis of anti-His antibody cross-reactivity of the proteins separated by SDS-PAGE. The lanes are the same as described for SDS-PAGE in panel A.</p

The PLS-DA analysis performed on quantified proteins from fiber standards.

<p>(A) The PLS-DA scores plot shows good separation of samples from different species, and samples from different breads of the same species are clustered together. (B) The variable influence on projection (VIP) plot shows that protein P1-P28 (with VIP value > 1) make most contribution to the separation of three groups in (A). Twenty out of the twenty-eight proteins also make significant discrimination in the ANOVA test (<i>p</i><0.01) and they are marked with asterisks.</p

Phylogenetic analysis of the CcTrx1 protein compared with other known Trx1 proteins.

<p>The numbers on the nodes indicate percentage frequencies in 2000 bootstrap replications. The amino acid sequence from <i>E. coli</i> was used as the out-group. The common names, sizes as well as GenBank accession numbers of the selected Trx1 amino acid sequences are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097509#pone-0097509-t002" target="_blank">Table 2</a>.</p

Evaluation of candidate markers in species identification of fiber standards.

<p>(A) Evaluation of 24 candidate protein markers selected for fiber identification. None of them show satisfactory specificity and sensitivity across all fiber samples. (B) Evaluation of 65 candidate peptide markers selected for fiber qualification. Ten peptides (marked with a red star) show sufficient specificity and sensitivity across all fiber samples. Each line represents identification results of a specific protein/peptide across all samples. Fiber sample annotation is the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147044#pone.0147044.g001" target="_blank">Fig 1</a>, and the appending number refers to the number of replicate of this sample.</p

The quantitative proteomic strategy for fiber marker discovery and fiber proteome profiling results.

<p>(A) The schematic workflow of marker discovery and validation with combined untargeted and targeted proteomic strategies. Two replicates of a specific fiber sample (S1 and S2) were labeled with intermediate and heavy dimethyl tags, a fiber mixture from three species with a light dimethyl label served as reference (REF). The mixture of S1, S2 and REF was analyzed by nanoLC-MS/MS in IDA mode for fiber proteome profiling and marker discovery. Then peptide markers were validated and used for fiber quantification with the parallel reaction monitoring (PRM) approach. (B) Numbers of protein identification in cashmere, wool and yak fiber samples. The result shows large overlap of the fiber proteome among three species. (C) Percentage of keratin and KAPs identified in three types of fibers. Detailed GO classification for each identified protein is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147044#pone.0147044.s002" target="_blank">S1 Table</a>.</p

The deduced amino acid sequence of the CcTrx1 protein compared with the Trx1 proteins from other species.

<p>The accession numbers are from the GenBank database.</p