Comprehensive Spectral Library Generation

To generate a comprehensive spectral library, the pooled sample from each group was processed. The DDA data were processed using Proteome Discoverer (Thermo Scientific, Germany) software and searched against the human SwissProt database appended with the iRT fusion protein sequence (Biognosys). A maximum of two missed cleavages for trypsin was used, cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation deamination and +43 on Kn (carbamyl) were used as variable modifications. The parent and fragment ion mass tolerances were set to 10 ppm and 0.02 Da, respectively. The applied false discovery rate (FDR) cutoff was 0.01 at the protein level. The results were then imported into Spectronaut Pulsar (Biognosys, Switzerland) software to generate the library. Additionally, DIA data were imported into Spectronaut Pulsar software and searched against the human SwissProt database to generate the DIA library. The final library was generated by combining the DDA and DIA libraries of all the enrolled samples.

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Wang Z., Liu X., Wang W., Xu J., Sun H., Wei J., Yu Y., Zhao Y., Wang X., Liao Z., Sun W., Jia L, & Zhang Y. (2023). UPLC-MS based integrated plasma proteomic and metabolomic profiling of TSC-RAML and its relationship with everolimus treatment. Frontiers in Molecular Biosciences, 10, 1000248.

Publication 2023

Proteome Discoverer iRT fusion protein sequence Spectronaut Pulsar

Cleavages Cysteine Deamination Human Library Methionine Parent Protein Protein sequence Proteome Pulsar Tolerances Trypsin

Corresponding Organization : Capital Medical University

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Variable analysis

independent variables

Data processing software (Proteome Discoverer and Spectronaut Pulsar)
Database used for searching (human SwissProt database with iRT fusion protein sequence)

dependent variables

Identified proteins
Spectral library generated

control variables

Trypsin cleavage specificity (maximum of two missed cleavages)
Cysteine carbamidomethylation (fixed modification)
Methionine oxidation, deamination, and +43 on Kn (carbamyl) (variable modifications)
Parent and fragment ion mass tolerances (10 ppm and 0.02 Da, respectively)
False discovery rate (FDR) cutoff (0.01 at the protein level)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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