Htrms converter

All DIA files were extracted from home generated plasma library containing 73 466 precursors, 55 433 modified peptides and 5190 proteins using Spectronaut X (Biognosys). Raw MS data files were converted to HTRMS files with HTRMS converter (Biognosys). HTRMS files were imported to Spectronaut with default parameters with the decoy generation set to “mutated.” Cutoff of fold change > 1.3 or <0.3785 and Q < 0.05 (FDR‐corrected P value for t test) was set for differentially expressed proteins. Correlation and ROC analysis were carried out using R software version 3.5.3.

A total of 61 MS runs (14 DDA and 47 DIA) of the meniscal samples were converted to HTRMS format using HTRMS Converter (Biognosys AG, Switzerland) in order to decrease data analysis search time. A spectral library was created in Spectronaut X™ Pulsar (version 12.0.20491.15, Biognosys AG) using all DIA and DDA runs. Default settings (BGS factory settings) were used with additional modifications: cysteine carbamidomethylation as a fixed modification, and deamination, pyro-glutamic acid (N-term Glu to pyroglutamic acid), methionine oxidation, hydroxyproline and acetylation as variable modifications. The human protein database (20190416_UP152602_n20415) was used as the background proteome. A subsequent protein search was conducted in Spectronaut™ Pulsar using the recently created spectral library and the same human database as background proteome. Default settings were used for the search. Precursor quantitation was performed at MS2 level, and area under the curve was used as quantitation type. The top three peptides (proteotypic) for each protein were averaged to calculate protein abundance¹⁵ (link).

The peptides were analyzed by LC–MS operating in data-independent acquisition (DIA) mode. The LC was operated as above, except a different gradient elution was used. The concentration of mobile phase B was increased from 2 to 50% over 60 min, increased to 99% over 6 min, held at 99% for 3 min, and decreased to 2% over 2 min.
The MS was the same instrument as above but operated in DIA mode. Mass spectra were acquired using a collision energy of 35%, resolution of 30 000, maximum inject time of 54 ms, and automatic gain control target of 5 × 10⁴. Staggered isolation windows of 12 Da in the range of 400 to 1000 m/z were used.
Data were analyzed with Spectronaut 15 using the directDIA workflow and default settings. Peak area intensities were exported from Spectronaut. Quantitative and statistical analysis was performed, and processing protein peak areas were determined by the Spectronaut software. Prior to library-based analysis of the DIA data, the DIA raw files were converted into htrms files using the htrms converter (Biognosys). MS1 and MS2 data were centroided during conversion, and the other parameters were set to default. The htrms files were analyzed with Spectronaut (version: 15, Biognosys) via directDIA. Precursor and protein identifications were filtered to 1% false discovery rate.