Improving Protein Detection Confidence Using SWATH-Mass Spectrometry with Large Peptide Reference Libraries

Wu, Jemma X.; Pascovici, Dana; Ignjatović, Vera; Song, Xiaomin; Molloy, Mark P.

doi:10.1002/pmic.201700174

Cited by 8 publications

(8 citation statements)

References 20 publications

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“…Two different FDR criteria were used for the SWATH data extracted using the two different (rPSL and plasma) libraries. For the SWATH data extracted using the merged spectral library, the default PeakView FDR criterion was used, i.e., peptides with at least one sample satisfying FDR < 0.01 were kept. , For the SWATH data extracted using the rPSL, a more stringent FDR criterion (≥3 replicates within a group having FDR < 0.01) was applied due to the potential for less effective FDR calculation resulting from the use of a small spectral library.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…Some have used a range of protein/peptide fractionation strategies (e.g., HAP depletion and/or combinations of peptide fractionation methods) prior to DDA experiments. , Other researchers have generated larger libraries by combining two or more existing DDA libraries . Equally, software tools have been developed that facilitate library concatenation. , Clearly, generation of comprehensive, high-quality peptide spectral libraries for high-quality SWATH/DIA analysis is crucial, as these represent the biological/proteome space of biospecimens being interrogated.…”

Section: Introductionmentioning

confidence: 99%

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Use of a Recombinant Biomarker Protein DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

et al. 2021

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Credible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). Here, we employed a mixture of recombinant proteins in DDA libraries to subsequently detect cancer-associated low abundance plasma proteins using SWATH/DIA.The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 human recombinant proteins that had been previously implicated as possible cancer biomarkers in both our own and other studies. The rPSL was then used to identify proteins from non-depleted colorectal cancer (CRC) plasmas by SWATH-MS. Most (32/36) of the proteins in the rPSL were reliably identified in plasma samples, including 8 proteins (BTC, CXCL10, IL1B, IL6, ITGB6, TGFα, TNF, TP53) not previously detected using highstringency MS in human plasmas according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and post-digestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 were identified using DDA-MS. The 20 additional proteins exclusively identified by using the rPSL approach with SWATH were mostly lower abundance (i.e., <10ng/ml) plasma proteins. To mitigate FDR concerns, and replicating a more typical approach, the DDA rPSL was also merged into a human plasma DDA library.When SWATH identification was repeated using this merged library, the majority (33/36) of low abundance plasma proteins from the rPSL could still be identified using high-stringency HPP Guidelines v3.0 protein inference criteria.Colorectal

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Section: Materials and Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Use of a Recombinant Biomarker Protein DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

et al. 2021

Self Cite

View full text Add to dashboard Cite

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“…Two different FDR criteria were used for the SWATH data extracted using the two different (rPSL and plasma) libraries. For the SWATH data extracted using the merged spectral library, the default PeakView FDR criterion was used, i.e., peptides with at least one sample satisfying FDR < 0.01 were kept (15, 21). For the SWATH data extracted using the rPSL, a more stringent FDR criterion (>3 replicates within a group having FDR < 0.01) was applied due to the potential for less effective FDR calculation resulting from the use of a small spectral library.…”

Section: Methodsmentioning

confidence: 99%

“…Other researchers have generated larger libraries by combining 2 or more existing libraries (20). Equally, software tools have been developed that facilitate library concatenation (15, 21). Clearly, generation of comprehensive, high-quality peptide spectral libraries for high-quality SWATH/DIA analysis is crucial, as these represent the biological/proteome space of biospecimens being interrogated.…”

Section: Introductionmentioning

confidence: 99%

A Recombinant Protein Biomarker DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

Ahn

Kamath

Mohamedali

et al. 2020

Preprint

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Credible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). Here, we employed a mixture of recombinant proteins in DDA libraries to subsequently detect cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 human recombinant proteins that had been previously implicated as possible cancer biomarkers in both our own and other studies. The rPSL was then used to identify proteins from non-depleted colorectal cancer (CRC) plasmas by SWATH-MS. Most (32/36) of the proteins in the rPSL were reliably identified in plasma samples, including 8 proteins (BTC, CXCL10, IL1B, IL6, ITGB6, TGFα, TNF, TP53) not previously detected using high-stringency MS in human plasmas according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and post-digestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 were identified using DDA-MS. The 20 additional proteins exclusively identified by using the rPSL approach with SWATH were mostly lower abundance (i.e., <10ng/ml) plasma proteins. To mitigate FDR concerns, and replicating a more typical approach, the DDA rPSL was also merged into a human plasma DDA library. When SWATH identification was repeated using this merged library, the majority (33/36) of low abundance plasma proteins from the rPSL could still be identified using high-stringency HPP Guidelines v3.0 protein inference criteria.

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“…In the subsequent data analysis phase, the corresponding dataset is searched in a “triple quadrupole-like” mode, thus not considering the whole MS/MS scan spectrum, but by searching for several precursor to fragment transitions that identify and quantify the corresponding peptide. This search is made possible with the use of an ion library, previously acquired in a classical data dependent, full-spectrum mode (Fabre et al, 2017; Wu et al, 2017) [2] , [3] . The SWATH protocol, combining the protein identification power of high-resolution MS/MS spectra with the robustness and accuracy in analyte quantification of triple-quad targeted workflows, has become very popular in proteomics research.…”

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confidence: 99%

A new SWATH ion library for mouse adult hippocampal neural stem cells

et al. 2018

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Over the last years, the SWATH data-independent acquisition protocol (Sequential Window acquisition of All THeoretical mass spectra) has become a cornerstone for the worldwide proteomics community (Collins et al., 2017) [1]. In this approach, a high-resolution quadrupole-ToF mass spectrometer acquires thousands of MS/MS data by selecting not just a single precursor at a time, but by allowing a broad m/z range to be fragmented. This acquisition window is then sequentially moved from the lowest to the highest mass selection range. This technique enables the acquisition of thousands of high-resolution MS/MS spectra per minute in a standard LC–MS run. In the subsequent data analysis phase, the corresponding dataset is searched in a “triple quadrupole-like” mode, thus not considering the whole MS/MS scan spectrum, but by searching for several precursor to fragment transitions that identify and quantify the corresponding peptide. This search is made possible with the use of an ion library, previously acquired in a classical data dependent, full-spectrum mode (Fabre et al., 2017; Wu et al., 2017) [2], [3]. The SWATH protocol, combining the protein identification power of high-resolution MS/MS spectra with the robustness and accuracy in analyte quantification of triple-quad targeted workflows, has become very popular in proteomics research. The major drawback lies in the ion library itself, which is normally demanding and time-consuming to build. Conversely, through the realignment of chromatographic retention times, an ion library of a given proteome can relatively easily be tailored upon “any” proteomics experiment done on the same proteome. We are thus hereby sharing with the worldwide proteomics community our newly acquired ion library of mouse adult hippocampal neural stem cells. Given the growing effort in neuroscience research involving proteomics experiments (Pons-Espinal et al., 2017; Sarnyai and Guest, 2017; Sethi et al., 2015; Bramini et al., 2016) [4,[5], [6], [7], we believe that this data might be of great help for the neuroscience community. All the here reported data (RAW files, results and ion library) can be freely downloaded from the SWATHATLAS (Deutsch et al., 2008) [8] website (http://www.peptideatlas.org/PASS/PASS01110)

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Improving Protein Detection Confidence Using SWATH-Mass Spectrometry with Large Peptide Reference Libraries

Cited by 8 publications

References 20 publications

Use of a Recombinant Biomarker Protein DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

Use of a Recombinant Biomarker Protein DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

A Recombinant Protein Biomarker DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

A new SWATH ion library for mouse adult hippocampal neural stem cells

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