DIA-NN 1.8.1 (Data-Independent Acquisition by Neural Networks) Compiled on Apr 14 2022 15:31:19 Current date and time: Sat Feb 24 21:19:32 2024 CPU: GenuineIntel Intel(R) Xeon(R) W-2225 CPU @ 4.10GHz SIMD instructions: AVX AVX2 AVX512CD AVX512F FMA SSE4.1 SSE4.2 Logical CPU cores: 8 diann.exe --f M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K1.raw --f M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K2.raw --f M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K3.raw --f M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K4.raw --f M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W1.raw --f M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W2.raw --f M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W3.raw --f M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W4.raw --lib --threads 4 --verbose 1 --out M:\Yu\OSU_Xin\report.tsv --qvalue 0.01 --matrices --predictor --fasta C:\Users\proteomics\Documents\FASTA database\uniprot-reviewed_yes+taxonomy_10090_mouse_reviewed_20230323.fasta --fasta-search --min-fr-mz 200 --max-fr-mz 1800 --met-excision --cut K*,R* --missed-cleavages 2 --min-pep-len 7 --max-pep-len 30 --min-pr-mz 300 --max-pr-mz 1800 --min-pr-charge 1 --max-pr-charge 4 --unimod4 --var-mods 3 --var-mod UniMod:35,15.994915,M --mass-acc 20 --mass-acc-ms1 10.0 --reanalyse --relaxed-prot-inf --smart-profiling --peak-center --no-ifs-removal Thread number set to 4 Output will be filtered at 0.01 FDR Precursor/protein x samples expression level matrices will be saved along with the main report Deep learning will be used to generate a new in silico spectral library from peptides provided Library-free search enabled Min fragment m/z set to 200 Max fragment m/z set to 1800 N-terminal methionine excision enabled In silico digest will involve cuts at K*,R* Maximum number of missed cleavages set to 2 Min peptide length set to 7 Max peptide length set to 30 Min precursor m/z set to 300 Max precursor m/z set to 1800 Min precursor charge set to 1 Max precursor charge set to 4 Cysteine carbamidomethylation enabled as a fixed modification Maximum number of variable modifications set to 3 Modification UniMod:35 with mass delta 15.9949 at M will be considered as variable A spectral library will be created from the DIA runs and used to reanalyse them; .quant files will only be saved to disk during the first step Highly heuristic protein grouping will be used, to reduce the number of protein groups obtained; this mode is recommended for benchmarking protein ID numbers; use with caution for anything else When generating a spectral library, in silico predicted spectra will be retained if deemed more reliable than experimental ones Fixed-width center of each elution peak will be used for quantification Interference removal from fragment elution curves disabled Mass accuracy will be fixed to 2e-05 (MS2) and 1e-05 (MS1) Exclusion of fragments shared between heavy and light peptides from quantification is not supported in FASTA digest mode - disabled; to enable, generate an in silico predicted spectral library and analyse with this library 8 files will be processed [0:00] Loading FASTA C:\Users\proteomics\Documents\FASTA database\uniprot-reviewed_yes+taxonomy_10090_mouse_reviewed_20230323.fasta [0:11] Processing FASTA [0:27] Assembling elution groups [0:39] 8384811 precursors generated [0:39] Gene names missing for some isoforms [0:39] Library contains 17130 proteins, and 16773 genes [0:39] [0:58] [81:00] [97:16] [98:05] [98:12] Saving the library to lib.predicted.speclib [98:24] Initialising library [98:30] First pass: generating a spectral library from DIA data [98:30] File #1/8 [98:30] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K1.raw [99:10] 5145112 library precursors are potentially detectable [99:11] Processing... [99:24] Cannot perform MS1 mass calibration, too few confidently identified precursors [100:45] RT window set to 12.2701 [100:45] Peak width: 2.808 [100:45] Scan window radius set to 6 [100:46] Recommended MS1 mass accuracy setting: 9.80433 ppm [123:24] Removing low confidence identifications [123:24] Removing interfering precursors [123:37] Training neural networks: 189310 targets, 145682 decoys [124:06] Number of IDs at 0.01 FDR: 108840 [124:07] Calculating protein q-values [124:08] Number of genes identified at 1% FDR: 7869 (precursor-level), 7120 (protein-level) (inference performed using proteotypic peptides only) [124:08] Quantification [124:36] Quantification information saved to M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K1.raw.quant. [124:37] File #2/8 [124:37] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K2.raw [125:19] 5145112 library precursors are potentially detectable [125:20] Processing... [125:41] Cannot perform MS1 mass calibration, too few confidently identified precursors [128:00] RT window set to 12.3578 [128:01] Recommended MS1 mass accuracy setting: 22.2054 ppm [150:17] Removing low confidence identifications [150:18] Removing interfering precursors [150:30] Training neural networks: 188171 targets, 145665 decoys [150:59] Number of IDs at 0.01 FDR: 110135 [151:00] Calculating protein q-values [151:01] Number of genes identified at 1% FDR: 7915 (precursor-level), 7166 (protein-level) (inference performed using proteotypic peptides only) [151:01] Quantification [151:36] Quantification information saved to M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K2.raw.quant. [151:37] File #3/8 [151:37] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K3.raw [152:19] 5145112 library precursors are potentially detectable [152:20] Processing... [152:36] Cannot perform MS1 mass calibration, too few confidently identified precursors [153:57] RT window set to 12.369 [153:57] Recommended MS1 mass accuracy setting: 14.9039 ppm [176:25] Removing low confidence identifications [176:26] Removing interfering precursors [176:40] Training neural networks: 184846 targets, 141433 decoys [177:10] Number of IDs at 0.01 FDR: 108142 [177:11] Calculating protein q-values [177:12] Number of genes identified at 1% FDR: 7952 (precursor-level), 7212 (protein-level) (inference performed using proteotypic peptides only) [177:12] Quantification [177:36] Quantification information saved to M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K3.raw.quant. [177:37] File #4/8 [177:37] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K4.raw [178:15] 5145112 library precursors are potentially detectable [178:15] Processing... [178:36] Cannot perform MS1 mass calibration, too few confidently identified precursors [180:07] RT window set to 12.3468 [180:08] Recommended MS1 mass accuracy setting: 10.8572 ppm [199:33] Removing low confidence identifications [199:33] Removing interfering precursors [199:45] Training neural networks: 160001 targets, 125361 decoys [200:09] Number of IDs at 0.01 FDR: 95885 [200:10] Calculating protein q-values [200:11] Number of genes identified at 1% FDR: 7537 (precursor-level), 6883 (protein-level) (inference performed using proteotypic peptides only) [200:11] Quantification [200:36] Quantification information saved to M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K4.raw.quant. [200:37] File #5/8 [200:37] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W1.raw [201:20] 5145112 library precursors are potentially detectable [201:21] Processing... [201:43] Cannot perform MS1 mass calibration, too few confidently identified precursors [203:20] RT window set to 11.5174 [203:21] Recommended MS1 mass accuracy setting: 18.0537 ppm [224:26] Removing low confidence identifications [224:26] Removing interfering precursors [224:38] Training neural networks: 179352 targets, 139670 decoys [225:06] Number of IDs at 0.01 FDR: 107495 [225:07] Calculating protein q-values [225:08] Number of genes identified at 1% FDR: 7879 (precursor-level), 7132 (protein-level) (inference performed using proteotypic peptides only) [225:08] Quantification [225:36] Quantification information saved to M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W1.raw.quant. [225:37] File #6/8 [225:37] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W2.raw [226:19] 5145112 library precursors are potentially detectable [226:20] Processing... [226:42] Cannot perform MS1 mass calibration, too few confidently identified precursors [228:19] RT window set to 11.9714 [228:19] Recommended MS1 mass accuracy setting: 18.6096 ppm [249:40] Removing low confidence identifications [249:40] Removing interfering precursors [249:53] Training neural networks: 182562 targets, 140647 decoys [250:20] Number of IDs at 0.01 FDR: 108059 [250:22] Calculating protein q-values [250:22] Number of genes identified at 1% FDR: 7831 (precursor-level), 7070 (protein-level) (inference performed using proteotypic peptides only) [250:22] Quantification [250:38] Quantification information saved to M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W2.raw.quant. [250:40] File #7/8 [250:40] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W3.raw [251:24] 5145112 library precursors are potentially detectable [251:25] Processing... [251:39] Cannot perform MS1 mass calibration, too few confidently identified precursors [253:18] RT window set to 12.553 [253:18] Recommended MS1 mass accuracy setting: 14.5871 ppm [276:30] Removing low confidence identifications [276:30] Removing interfering precursors [276:42] Training neural networks: 174201 targets, 134829 decoys [277:09] Number of IDs at 0.01 FDR: 103779 [277:10] Calculating protein q-values [277:11] Number of genes identified at 1% FDR: 7727 (precursor-level), 6986 (protein-level) (inference performed using proteotypic peptides only) [277:11] Quantification [277:37] Quantification information saved to M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W3.raw.quant. [277:39] File #8/8 [277:39] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W4.raw [278:23] 5145112 library precursors are potentially detectable [278:24] Processing... [278:46] Cannot perform MS1 mass calibration, too few confidently identified precursors [280:25] RT window set to 12.4371 [280:25] Recommended MS1 mass accuracy setting: 17.0391 ppm [303:32] Removing low confidence identifications [303:33] Removing interfering precursors [303:45] Training neural networks: 175395 targets, 134660 decoys [304:12] Number of IDs at 0.01 FDR: 104907 [304:13] Calculating protein q-values [304:14] Number of genes identified at 1% FDR: 7746 (precursor-level), 7090 (protein-level) (inference performed using proteotypic peptides only) [304:14] Quantification [304:37] Quantification information saved to M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W4.raw.quant. [304:38] Cross-run analysis [304:38] Reading quantification information: 8 files [304:47] Quantifying peptides [304:56] Assembling protein groups [305:01] Quantifying proteins [305:02] Calculating q-values for protein and gene groups [305:03] Calculating global q-values for protein and gene groups [305:04] Writing report [309:36] Report saved to M:\Yu\OSU_Xin\report-first-pass.tsv. [309:36] Saving precursor levels matrix [309:40] Precursor levels matrix (1% precursor and protein group FDR) saved to M:\Yu\OSU_Xin\report-first-pass.pr_matrix.tsv. [309:40] Saving protein group levels matrix [309:41] Protein group levels matrix (1% precursor FDR and protein group FDR) saved to M:\Yu\OSU_Xin\report-first-pass.pg_matrix.tsv. [309:41] Saving gene group levels matrix [309:41] Gene groups levels matrix (1% precursor FDR and protein group FDR) saved to M:\Yu\OSU_Xin\report-first-pass.gg_matrix.tsv. [309:41] Saving unique genes levels matrix [309:41] Unique genes levels matrix (1% precursor FDR and protein group FDR) saved to M:\Yu\OSU_Xin\report-first-pass.unique_genes_matrix.tsv. [309:41] Stats report saved to M:\Yu\OSU_Xin\report-first-pass.stats.tsv [309:41] Generating spectral library: [309:41] 146949 precursors passing the FDR threshold are to be extracted [309:41] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K1.raw [310:26] 5145112 library precursors are potentially detectable [310:28] 15591 spectra added to the library [310:29] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K2.raw [311:12] 5145112 library precursors are potentially detectable [311:13] 13206 spectra added to the library [311:14] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K3.raw [311:57] 5145112 library precursors are potentially detectable [311:59] 15803 spectra added to the library [312:00] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K4.raw [312:41] 5145112 library precursors are potentially detectable [312:43] 19145 spectra added to the library [312:43] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W1.raw [313:29] 5145112 library precursors are potentially detectable [313:31] 15309 spectra added to the library [313:32] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W2.raw [314:14] 5145112 library precursors are potentially detectable [314:15] 10440 spectra added to the library [314:16] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W3.raw [315:00] 5145112 library precursors are potentially detectable [315:01] 9949 spectra added to the library [315:02] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W4.raw [315:48] 5145112 library precursors are potentially detectable [315:50] 11463 spectra added to the library [315:50] Saving spectral library to lib.tsv [316:06] 146949 precursors saved [316:06] Loading the generated library and saving it in the .speclib format [316:06] Loading spectral library lib.tsv [316:13] Spectral library loaded: 9544 protein isoforms, 10326 protein groups and 146949 precursors in 120136 elution groups. [316:13] Loading protein annotations from FASTA C:\Users\proteomics\Documents\FASTA database\uniprot-reviewed_yes+taxonomy_10090_mouse_reviewed_20230323.fasta [316:13] Gene names missing for some isoforms [316:13] Library contains 9544 proteins, and 9390 genes [316:13] Saving the library to lib.tsv.speclib [316:20] Second pass: using the newly created spectral library to reanalyse the data [316:20] File #1/8 [316:20] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K1.raw [316:58] 146949 library precursors are potentially detectable [316:58] Processing... [317:01] RT window set to 3.07305 [317:01] Recommended MS1 mass accuracy setting: 20.4128 ppm [317:16] Removing low confidence identifications [317:16] Removing interfering precursors [317:21] Training neural networks: 138269 targets, 116062 decoys [317:41] Number of IDs at 0.01 FDR: 123971 [317:43] Calculating protein q-values [317:43] Number of genes identified at 1% FDR: 8223 (precursor-level), 7521 (protein-level) (inference performed using proteotypic peptides only) [317:43] Quantification [317:45] File #2/8 [317:45] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K2.raw [318:27] 146949 library precursors are potentially detectable [318:27] Processing... [318:29] RT window set to 3.08053 [318:29] Recommended MS1 mass accuracy setting: 17.4807 ppm [318:44] Removing low confidence identifications [318:44] Removing interfering precursors [318:49] Training neural networks: 138079 targets, 113691 decoys [319:10] Number of IDs at 0.01 FDR: 126707 [319:12] Calculating protein q-values [319:12] Number of genes identified at 1% FDR: 8337 (precursor-level), 7563 (protein-level) (inference performed using proteotypic peptides only) [319:12] Quantification [319:14] File #3/8 [319:14] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K3.raw [319:51] 146949 library precursors are potentially detectable [319:51] Processing... [319:54] RT window set to 3.07488 [319:54] Recommended MS1 mass accuracy setting: 24.8109 ppm [320:08] Removing low confidence identifications [320:08] Removing interfering precursors [320:13] Training neural networks: 137529 targets, 112621 decoys [320:34] Number of IDs at 0.01 FDR: 125119 [320:35] Calculating protein q-values [320:35] Number of genes identified at 1% FDR: 8335 (precursor-level), 7574 (protein-level) (inference performed using proteotypic peptides only) [320:35] Quantification [320:38] File #4/8 [320:38] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_K4.raw [321:11] 146949 library precursors are potentially detectable [321:11] Processing... [321:15] RT window set to 2.98742 [321:15] Recommended MS1 mass accuracy setting: 26.1927 ppm [321:28] Removing low confidence identifications [321:28] Removing interfering precursors [321:32] Training neural networks: 127904 targets, 102451 decoys [321:51] Number of IDs at 0.01 FDR: 109660 [321:52] Calculating protein q-values [321:52] Number of genes identified at 1% FDR: 8006 (precursor-level), 7282 (protein-level) (inference performed using proteotypic peptides only) [321:52] Quantification [321:54] File #5/8 [321:54] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W1.raw [322:38] 146949 library precursors are potentially detectable [322:38] Processing... [322:40] RT window set to 3.08789 [322:40] Recommended MS1 mass accuracy setting: 29.3085 ppm [322:55] Removing low confidence identifications [322:55] Removing interfering precursors [323:00] Training neural networks: 138855 targets, 115832 decoys [323:21] Number of IDs at 0.01 FDR: 127025 [323:22] Calculating protein q-values [323:23] Number of genes identified at 1% FDR: 8293 (precursor-level), 7455 (protein-level) (inference performed using proteotypic peptides only) [323:23] Quantification [323:25] File #6/8 [323:25] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W2.raw [324:00] 146949 library precursors are potentially detectable [324:00] Processing... [324:02] RT window set to 3.08456 [324:02] Recommended MS1 mass accuracy setting: 19.7106 ppm [324:17] Removing low confidence identifications [324:17] Removing interfering precursors [324:22] Training neural networks: 137098 targets, 113509 decoys [324:42] Number of IDs at 0.01 FDR: 124930 [324:44] Calculating protein q-values [324:44] Number of genes identified at 1% FDR: 8271 (precursor-level), 7453 (protein-level) (inference performed using proteotypic peptides only) [324:44] Quantification [324:46] File #7/8 [324:46] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W3.raw [325:27] 146949 library precursors are potentially detectable [325:27] Processing... [325:30] RT window set to 3.09416 [325:30] Recommended MS1 mass accuracy setting: 21.9213 ppm [325:44] Removing low confidence identifications [325:44] Removing interfering precursors [325:49] Training neural networks: 137286 targets, 115647 decoys [326:11] Number of IDs at 0.01 FDR: 122931 [326:12] Calculating protein q-values [326:12] Number of genes identified at 1% FDR: 8263 (precursor-level), 7492 (protein-level) (inference performed using proteotypic peptides only) [326:12] Quantification [326:15] File #8/8 [326:15] Loading run M:\Yu\OSU_Xin\20240225_OSU_TFA_E3_W4.raw [326:54] 146949 library precursors are potentially detectable [326:54] Processing... [326:57] RT window set to 3.05685 [326:57] Recommended MS1 mass accuracy setting: 16.4655 ppm [327:11] Removing low confidence identifications [327:11] Removing interfering precursors [327:16] Training neural networks: 136351 targets, 115413 decoys [327:36] Number of IDs at 0.01 FDR: 122043 [327:37] Calculating protein q-values [327:38] Number of genes identified at 1% FDR: 8224 (precursor-level), 7480 (protein-level) (inference performed using proteotypic peptides only) [327:38] Quantification [327:40] Cross-run analysis [327:40] Reading quantification information: 8 files [327:42] Quantifying peptides [327:51] Quantifying proteins [327:53] Calculating q-values for protein and gene groups [327:54] Calculating global q-values for protein and gene groups [327:54] Writing report [333:12] Report saved to M:\Yu\OSU_Xin\report.tsv. [333:12] Saving precursor levels matrix [333:30] Precursor levels matrix (1% precursor and protein group FDR) saved to M:\Yu\OSU_Xin\report.pr_matrix.tsv. [333:30] Saving protein group levels matrix [333:31] Protein group levels matrix (1% precursor FDR and protein group FDR) saved to M:\Yu\OSU_Xin\report.pg_matrix.tsv. [333:31] Saving gene group levels matrix [333:31] Gene groups levels matrix (1% precursor FDR and protein group FDR) saved to M:\Yu\OSU_Xin\report.gg_matrix.tsv. [333:31] Saving unique genes levels matrix [333:32] Unique genes levels matrix (1% precursor FDR and protein group FDR) saved to M:\Yu\OSU_Xin\report.unique_genes_matrix.tsv. [333:32] Stats report saved to M:\Yu\OSU_Xin\report.stats.tsv Finished