TopPIC Suite

1 Data and settings

  • Data: 480_F3_02.mzML, a top-down LC-MS/MS data file of a human cell lysate fraction with 2,803 MS1 and 4,323 MS/MS spectra (centroided). The data are available here.
  • Database: UniProt human reviewed proteins (SwissProt) with 20,380 protein sequences. TopPIC appends a shuffled decoy sequence for each protein for FDR estimation, so the searched database contains 20,380 target and 20,380 decoy sequences.
  • Database index: before the searches, the index of the protein database was generated with topindex -u 16 -f C57 -d uniprot-reviewed_yes+taxonomy_9606.fasta (TopPIC Suite 2.1.0; 2.9 minutes, 10.9 GB of memory). The index takes 93 GB of disk space. TopPIC 2.0.0 and 1.9.0 used an index generated with the same options in their own file layouts (172 GB and 201 GB).
  • Computer: a Linux workstation (Ubuntu 26.04.1 LTS) with an Intel Core i9-10900 CPU (10 cores, 20 threads, 2.80 GHz base clock, 5.20 GHz max turbo) and 62 GB of RAM. The data, the protein database index, and the output files were stored on a 1 TB Crucial T500 NVMe SSD (connected through PCIe 3.0 x4).

All searches used carbamidomethylation on cysteine as a fixed modification and report identifications at 1% spectrum-level and 1% proteoform-level FDR.

2 TopPIC Suite 1.9.0 vs 2.0.0 vs 2.1.0

This benchmark compares the identification results and the running times of TopFD and TopPIC in TopPIC Suite versions 1.9.0, 2.0.0, and 2.1.0 with their default settings: at most one unexpected mass shift and no variable modifications. All runs used 16 threads.

Settings

Version 1.9.0Version 2.0.0Version 2.1.0
TopFD commandtopfd -u 16 480_F3_02.mzMLtopfd -u 16 480_F3_02.mzMLtopfd -u 16 480_F3_02.mzML
TopPIC commandtoppic -u 16 -f C57 -d -t FDR -T FDR -y FDR db.fasta 480_F3_02_ms2.msaligntoppic -u 16 -f C57 -d -t FDR -T FDR -y FDR db.fasta 480_F3_02_ms2.msaligntoppic -u 16 -f C57 -d -t FDR -T FDR -y FDR db.fasta 480_F3_02_ms2.msalign

All three versions write an SQLite file with the centroided spectra in TopFD, which TopPIC uses for post mass matching (benchmark 4). TopPIC 1.9.0 and 2.0.0 store the identifications in that file; TopPIC 2.1.0 stores them in a file of its own (480_F3_02_toppic.sqlite), and TopFD and TopPIC 2.1.0 compress their SQLite files (843 MB to 295 MB for TopFD). Post mass matching, storing the identifications, and the compression are included in the running times.

Results

Identifications at 1% FDR Identifications at 1% FDR 1.9.0 2.0.0 2.1.0 0 1,000 2,000 3,000 4,000 Number of identifications PrSMs, Version 1.9.0: 2,870 2,870 PrSMs, Version 2.0.0: 2,966 2,966 PrSMs, Version 2.1.0: 3,207 3,207 PrSMs Proteoforms, Version 1.9.0: 1,686 1,686 Proteoforms, Version 2.0.0: 1,765 1,765 Proteoforms, Version 2.1.0: 1,918 1,918 Proteoforms
Running time of TopFD Running time of TopFD 0 5 10 15 Wall-clock time (minutes) Version 1.9.0, TopFD: 12.6 minutes 12.6 1.9.0 Version 2.0.0, TopFD: 5.7 minutes 5.7 2.0.0 Version 2.1.0, TopFD: 6.9 minutes 6.9 2.1.0
Running time of TopPIC Running time of TopPIC 0 20 40 60 80 Wall-clock time (minutes) Version 1.9.0, TopPIC: 57.4 minutes 57.4 1.9.0 Version 2.0.0, TopPIC: 6.2 minutes 6.2 2.0.0 Version 2.1.0, TopPIC: 5.3 minutes 5.3 2.1.0
Version 1.9.0Version 2.0.0Version 2.1.0
PrSMs (1% spectrum-level FDR)2,8702,966 (+3.3%)3,207 (+8.1%)
Proteoforms (1% proteoform-level FDR)1,6861,765 (+4.7%)1,918 (+8.7%)
Proteins (1% protein-level FDR)393416436
TopFD running time (minutes)12.65.7 (2.2X speedup)6.9 (21% longer)
TopPIC running time (minutes)57.46.2 (9.2X speedup)5.3 (1.17X speedup)

Changes and speedups in parentheses are relative to the previous version: 1.9.0 for version 2.0.0 and 2.0.0 for version 2.1.0.

Version 2.1.0 identified 8.1% more PrSMs and 8.7% more proteoforms than version 2.0.0, and 11.7% more PrSMs and 13.8% more proteoforms than version 1.9.0. TopFD 2.1.0 took 6.9 minutes instead of 5.7, because it spends 77 seconds on three new steps: it prepares its SQLite file for 3D visualization in TopMSV, compacts it, and compresses it. The rest took 5.6 minutes. TopPIC 2.1.0 ran 1.17 times faster than version 2.0.0 and 10.9 times faster than version 1.9.0.

3 TopPIC 2.1.0 search settings

This benchmark compares seven search settings of TopPIC 2.1.0 on the same data set, database, and computer as benchmark 2: the default setting (at most one unexpected mass shift and no variable modifications), searches with variable modifications (-b) from two files, methionine oxidation only (var_mods.txt) and four common modifications (var_4_mods.txt), searches with up to two unexpected mass shifts (-s 2) alone and with each file, and the search with two shifts and the four modifications with its FDRs computed from a rescore of the PrSMs (-q). All seven searches used the same TopFD 2.1.0 output, 16 threads, and 1% FDR at the spectrum, proteoform, and protein levels.

Settings

SettingTopPIC options (in addition to -u 16 -f C57 -d -t FDR -T FDR -y FDR)Search space
adefaultAt most 1 unexpected mass shift, no variable modification
b-b var_mods.txtAt most 1 unexpected mass shift, variable oxidation on M (at most 3 sites)
c-b var_4_mods.txtAt most 1 unexpected mass shift, four variable modifications (at most 3 sites)
d-s 2At most 2 unexpected mass shifts
e-s 2 -b var_mods.txtAt most 2 unexpected mass shifts, variable oxidation on M
f-s 2 -b var_4_mods.txtAt most 2 unexpected mass shifts, four variable modifications
g-s 2 -b var_4_mods.txt -qSetting f with the FDRs computed from a rescore of the PrSMs

The file var_mods.txt defines a single variable modification: oxidation (+15.994915 Da) on methionine (Unimod 35). The file var_4_mods.txt defines four: oxidation on M, acetylation (+42.010565 Da) on K (Unimod 1), methylation (+14.015650 Da) on K and R (Unimod 34), and phosphorylation (+79.966331 Da) on S, T, and Y (Unimod 21). Both files are in the benchmark data. With -q (--rescore, off by default, new in version 2.1.0), TopPIC gives the best PrSM of every spectrum a score from a linear support vector machine trained on the target and decoy PrSMs with 3-fold cross-validation and ranks the PrSMs by it in the FDR computation. However, note that the FDRs computed from the rescore may be underestimated.

Results

Running time of TopPIC Running time of TopPIC No PTM or oxidation Four PTMs Four PTMs, -q 0 5 10 15 Wall-clock time (minutes) a: default, TopPIC: 5.3 minutes 5.3 a b: -b var_mods.txt, TopPIC: 5.7 minutes 5.7 b c: -b var_4_mods.txt, TopPIC: 6.9 minutes 6.9 c d: -s 2, TopPIC: 10.3 minutes 10.3 d e: -s 2 -b var_mods.txt, TopPIC: 10.7 minutes 10.7 e f: -s 2 -b var_4_mods.txt, TopPIC: 11.6 minutes 11.6 f g: -s 2 -b var_4_mods.txt -q, TopPIC: 12.7 minutes 12.7 g
Identifications at 1% FDR Identifications at 1% FDR PrSMs Proteoforms 0 1,000 2,000 3,000 4,000 Number of identifications 0% 20% 40% 60% 80% Spectral ID rate a: default, PrSMs: 3,207 (74.2% of the 4,323 MS/MS spectra) 3,207 a: default, Proteoforms: 1,918 1,918 a b: -b var_mods.txt, PrSMs: 3,251 (75.2% of the 4,323 MS/MS spectra) 3,251 b: -b var_mods.txt, Proteoforms: 1,943 1,943 b c: -b var_4_mods.txt, PrSMs: 3,305 (76.5% of the 4,323 MS/MS spectra) 3,305 c: -b var_4_mods.txt, Proteoforms: 1,960 1,960 c d: -s 2, PrSMs: 3,337 (77.2% of the 4,323 MS/MS spectra) 3,337 d: -s 2, Proteoforms: 1,988 1,988 d e: -s 2 -b var_mods.txt, PrSMs: 3,374 (78.0% of the 4,323 MS/MS spectra) 3,374 e: -s 2 -b var_mods.txt, Proteoforms: 2,004 2,004 e f: -s 2 -b var_4_mods.txt, PrSMs: 3,415 (79.0% of the 4,323 MS/MS spectra) 3,415 f: -s 2 -b var_4_mods.txt, Proteoforms: 2,023 2,023 f g: -s 2 -b var_4_mods.txt -q, PrSMs: 3,513 (81.3% of the 4,323 MS/MS spectra) 3,513 g: -s 2 -b var_4_mods.txt -q, Proteoforms: 2,063 2,063 g
SettingRunning time (minutes)Peak memoryPrSMsProteoformsProteins
a: default5.314.3 GB3,2071,918436
b: -b var_mods.txt5.717.5 GB3,251 (+1.4%)1,943 (+1.3%)437
c: -b var_4_mods.txt6.917.7 GB3,305 (+3.1%)1,960 (+2.2%)442
d: -s 210.313.7 GB3,337 (+4.1%)1,988 (+3.6%)440
e: -s 2 -b var_mods.txt10.717.5 GB3,374 (+5.2%)2,004 (+4.5%)440
f: -s 2 -b var_4_mods.txt11.617.8 GB3,415 (+6.5%)2,023 (+5.5%)445
g: -s 2 -b var_4_mods.txt -q12.717.7 GB3,513 (+9.5%)2,063 (+7.6%)452

Running time

Variable oxidation (-b var_mods.txt) adds 0.4 minutes to the default search and the four variable modifications (-b var_4_mods.txt) add 1.6 minutes; both need at most 3.4 GB more memory. Allowing two unexpected mass shifts (-s 2) is the expensive option: it adds a multi-shift filtering step and a two-shift search, and the running time grows 2.0 times, from 5.3 to 10.3 minutes. With both options, the search takes 10.7 minutes with oxidation and 11.6 minutes with the four modifications. The rescore (-q) takes 6 seconds.

Identifications

Setting g, the search of setting f with its FDRs computed from the rescore, gives the largest set: 3,513 PrSMs, 2,063 proteoforms, and 452 proteins, 9.5% more PrSMs and 7.6% more proteoforms than the default setting and 2.9% more PrSMs and 2.0% more proteoforms than setting f.

The four variable modifications identify 54 more PrSMs than oxidation alone with one unexpected mass shift (setting c against b) and 41 more with two (setting f against e). The phosphorylation, methylation, and acetylation sites are assigned by mass: a shift of +79.966 Da on S, T, or Y is reported as phosphorylation, so the fragment ions that localize each site should be checked before a site is accepted.

4 TopPIC with post mass matching

TopPIC matches theoretical fragment masses of a proteoform to the masses reported by TopFD, which requires a valid isotopic envelope with several peaks. Post mass matching matches theoretical fragment ions directly to centroided peaks to increase the number of matched fragment masses in identified PrSMs. It is enabled by default and can be turned off with --disable-post-match (-D). After the database search and E-value computation, each theoretical fragment mass of the best PrSM of a spectrum that no deconvoluted mass matched is searched in the centroided MS/MS spectrum. A fragment is accepted when its most abundant isotopic peak is present, at least --post-min-peak-num isotopic peaks are present (default 1), and the observed isotopic intensities fit the theoretical envelope. The added masses are scored with EnvCNN and used by all later steps. Post mass matching runs after the E-values and the proteoform clusters are final and changes neither.

Settings

Without post mass matchingWith post mass matching
TopFD commandtopfd -u 16 480_F3_02.mzML (one run for both searches)
TopPIC commandtoppic -u 16 -f C57 -d -t FDR -T FDR -y FDR -D db.fasta 480_F3_02_ms2.msaligntoppic -u 16 -f C57 -d -t FDR -T FDR -y FDR db.fasta 480_F3_02_ms2.msalign

Both runs searched the same TopFD 2.1.0 output with the default search settings of TopPIC 2.1.0; the run with post mass matching is setting a of benchmark 3.

Results

Distribution of matched fragment masses per PrSM Matched fragment masses per PrSM Without post mass matching With post mass matching 0 100 200 300 400 500 Number of PrSMs 0–4 matched masses, without post mass matching: 4 PrSMs 0–4 matched masses, with post mass matching: 1 PrSMs 0–4 5–9 matched masses, without post mass matching: 183 PrSMs 5–9 matched masses, with post mass matching: 49 PrSMs 10–14 matched masses, without post mass matching: 337 PrSMs 10–14 matched masses, with post mass matching: 150 PrSMs 10–14 15–19 matched masses, without post mass matching: 398 PrSMs 15–19 matched masses, with post mass matching: 288 PrSMs 20–24 matched masses, without post mass matching: 377 PrSMs 20–24 matched masses, with post mass matching: 329 PrSMs 20–24 25–29 matched masses, without post mass matching: 348 PrSMs 25–29 matched masses, with post mass matching: 327 PrSMs 30–34 matched masses, without post mass matching: 315 PrSMs 30–34 matched masses, with post mass matching: 364 PrSMs 30–34 35–39 matched masses, without post mass matching: 262 PrSMs 35–39 matched masses, with post mass matching: 314 PrSMs 40–44 matched masses, without post mass matching: 243 PrSMs 40–44 matched masses, with post mass matching: 294 PrSMs 40–44 45–49 matched masses, without post mass matching: 191 PrSMs 45–49 matched masses, with post mass matching: 277 PrSMs 50–54 matched masses, without post mass matching: 137 PrSMs 50–54 matched masses, with post mass matching: 195 PrSMs 50–54 55–59 matched masses, without post mass matching: 113 PrSMs 55–59 matched masses, with post mass matching: 157 PrSMs 60–64 matched masses, without post mass matching: 92 PrSMs 60–64 matched masses, with post mass matching: 131 PrSMs 60–64 65–69 matched masses, without post mass matching: 60 PrSMs 65–69 matched masses, with post mass matching: 91 PrSMs 70–74 matched masses, without post mass matching: 52 PrSMs 70–74 matched masses, with post mass matching: 81 PrSMs 70–74 75–79 matched masses, without post mass matching: 26 PrSMs 75–79 matched masses, with post mass matching: 56 PrSMs 80–84 matched masses, without post mass matching: 18 PrSMs 80–84 matched masses, with post mass matching: 29 PrSMs 80–84 85–89 matched masses, without post mass matching: 21 PrSMs 85–89 matched masses, with post mass matching: 19 PrSMs 90–94 matched masses, without post mass matching: 12 PrSMs 90–94 matched masses, with post mass matching: 20 PrSMs 90–94 95–99 matched masses, without post mass matching: 7 PrSMs 95–99 matched masses, with post mass matching: 11 PrSMs 100–104 matched masses, without post mass matching: 2 PrSMs 100–104 matched masses, with post mass matching: 10 PrSMs 100–104 105–109 matched masses, without post mass matching: 3 PrSMs 105–109 matched masses, with post mass matching: 6 PrSMs 110–114 matched masses, without post mass matching: 4 PrSMs 110–114 matched masses, with post mass matching: 2 PrSMs 110–114 115–119 matched masses, with post mass matching: 2 PrSMs 120–124 matched masses, without post mass matching: 2 PrSMs 120–124 matched masses, with post mass matching: 2 PrSMs 120–124 125–129 matched masses, with post mass matching: 1 PrSMs 130–134 matched masses, with post mass matching: 1 PrSMs 130–134 Matched fragment masses per PrSM
Matched fragment masses per PrSM, with vs without post mass matching Matched fragment masses per PrSM 0 0 20 20 40 40 60 60 80 80 100 100 120 120 140 140 y = x Without: 4, with: 4 (1 PrSM) Without: 4, with: 5 (2 PrSMs) Without: 4, with: 8 (1 PrSM) Without: 5, with: 5 (4 PrSMs) Without: 5, with: 6 (3 PrSMs) Without: 5, with: 7 (1 PrSM) Without: 5, with: 8 (1 PrSM) Without: 5, with: 9 (2 PrSMs) Without: 5, with: 11 (1 PrSM) Without: 5, with: 13 (1 PrSM) Without: 5, with: 14 (1 PrSM) Without: 6, with: 6 (4 PrSMs) Without: 6, with: 7 (6 PrSMs) Without: 6, with: 9 (6 PrSMs) Without: 6, with: 10 (1 PrSM) Without: 6, with: 11 (1 PrSM) Without: 6, with: 12 (1 PrSM) Without: 6, with: 14 (1 PrSM) Without: 6, with: 15 (4 PrSMs) Without: 6, with: 16 (1 PrSM) Without: 7, with: 7 (4 PrSMs) Without: 7, with: 9 (6 PrSMs) Without: 7, with: 10 (3 PrSMs) Without: 7, with: 11 (5 PrSMs) Without: 7, with: 12 (3 PrSMs) Without: 7, with: 13 (2 PrSMs) Without: 7, with: 14 (4 PrSMs) Without: 7, with: 15 (3 PrSMs) Without: 7, with: 16 (3 PrSMs) Without: 7, with: 17 (1 PrSM) Without: 7, with: 18 (1 PrSM) Without: 8, with: 8 (1 PrSM) Without: 8, with: 9 (6 PrSMs) Without: 8, with: 10 (2 PrSMs) Without: 8, with: 11 (10 PrSMs) Without: 8, with: 12 (5 PrSMs) Without: 8, with: 13 (5 PrSMs) Without: 8, with: 14 (4 PrSMs) Without: 8, with: 15 (4 PrSMs) Without: 8, with: 16 (3 PrSMs) Without: 8, with: 17 (2 PrSMs) Without: 8, with: 19 (1 PrSM) Without: 8, with: 20 (2 PrSMs) Without: 9, with: 9 (2 PrSMs) Without: 9, with: 10 (6 PrSMs) Without: 9, with: 11 (13 PrSMs) Without: 9, with: 12 (5 PrSMs) Without: 9, with: 13 (12 PrSMs) Without: 9, with: 14 (5 PrSMs) Without: 9, with: 15 (7 PrSMs) Without: 9, with: 16 (4 PrSMs) Without: 9, with: 17 (2 PrSMs) Without: 9, with: 18 (3 PrSMs) Without: 9, with: 19 (3 PrSMs) Without: 9, with: 21 (1 PrSM) Without: 9, with: 22 (1 PrSM) Without: 10, with: 11 (4 PrSMs) Without: 10, with: 12 (4 PrSMs) Without: 10, with: 13 (9 PrSMs) Without: 10, with: 14 (8 PrSMs) Without: 10, with: 15 (18 PrSMs) Without: 10, with: 16 (1 PrSM) Without: 10, with: 17 (1 PrSM) Without: 10, with: 18 (5 PrSMs) Without: 10, with: 19 (5 PrSMs) Without: 10, with: 20 (3 PrSMs) Without: 10, with: 21 (1 PrSM) Without: 10, with: 23 (1 PrSM) Without: 10, with: 24 (2 PrSMs) Without: 11, with: 11 (1 PrSM) Without: 11, with: 12 (2 PrSMs) Without: 11, with: 13 (12 PrSMs) Without: 11, with: 14 (3 PrSMs) Without: 11, with: 15 (4 PrSMs) Without: 11, with: 16 (9 PrSMs) Without: 11, with: 17 (8 PrSMs) Without: 11, with: 18 (5 PrSMs) Without: 11, with: 19 (3 PrSMs) Without: 11, with: 20 (2 PrSMs) Without: 11, with: 21 (3 PrSMs) Without: 11, with: 22 (1 PrSM) Without: 11, with: 23 (1 PrSM) Without: 12, with: 13 (4 PrSMs) Without: 12, with: 14 (7 PrSMs) Without: 12, with: 15 (14 PrSMs) Without: 12, with: 16 (7 PrSMs) Without: 12, with: 17 (6 PrSMs) Without: 12, with: 18 (12 PrSMs) Without: 12, with: 19 (5 PrSMs) Without: 12, with: 20 (3 PrSMs) Without: 12, with: 21 (6 PrSMs) Without: 12, with: 22 (4 PrSMs) Without: 12, with: 23 (1 PrSM) Without: 12, with: 24 (1 PrSM) Without: 12, with: 25 (3 PrSMs) Without: 13, with: 13 (1 PrSM) Without: 13, with: 14 (4 PrSMs) Without: 13, with: 15 (2 PrSMs) Without: 13, with: 16 (7 PrSMs) Without: 13, with: 17 (10 PrSMs) Without: 13, with: 18 (10 PrSMs) Without: 13, with: 19 (10 PrSMs) Without: 13, with: 20 (8 PrSMs) Without: 13, with: 21 (4 PrSMs) Without: 13, with: 22 (2 PrSMs) Without: 13, with: 23 (1 PrSM) Without: 13, with: 24 (2 PrSMs) Without: 13, with: 25 (2 PrSMs) Without: 13, with: 26 (2 PrSMs) Without: 13, with: 28 (1 PrSM) Without: 13, with: 30 (1 PrSM) Without: 14, with: 15 (3 PrSMs) Without: 14, with: 16 (8 PrSMs) Without: 14, with: 17 (7 PrSMs) Without: 14, with: 18 (13 PrSMs) Without: 14, with: 19 (10 PrSMs) Without: 14, with: 20 (7 PrSMs) Without: 14, with: 21 (7 PrSMs) Without: 14, with: 22 (7 PrSMs) Without: 14, with: 23 (4 PrSMs) Without: 14, with: 24 (7 PrSMs) Without: 14, with: 25 (2 PrSMs) Without: 14, with: 26 (1 PrSM) Without: 14, with: 27 (2 PrSMs) Without: 14, with: 29 (2 PrSMs) Without: 14, with: 30 (1 PrSM) Without: 15, with: 16 (3 PrSMs) Without: 15, with: 17 (4 PrSMs) Without: 15, with: 18 (12 PrSMs) Without: 15, with: 19 (10 PrSMs) Without: 15, with: 20 (9 PrSMs) Without: 15, with: 21 (10 PrSMs) Without: 15, with: 22 (11 PrSMs) Without: 15, with: 23 (8 PrSMs) Without: 15, with: 24 (8 PrSMs) Without: 15, with: 25 (7 PrSMs) Without: 15, with: 26 (1 PrSM) Without: 15, with: 27 (1 PrSM) Without: 15, with: 30 (1 PrSM) Without: 15, with: 32 (1 PrSM) Without: 16, with: 16 (1 PrSM) Without: 16, with: 17 (3 PrSMs) Without: 16, with: 18 (4 PrSMs) Without: 16, with: 19 (7 PrSMs) Without: 16, with: 20 (7 PrSMs) Without: 16, with: 21 (6 PrSMs) Without: 16, with: 22 (7 PrSMs) Without: 16, with: 23 (5 PrSMs) Without: 16, with: 24 (5 PrSMs) Without: 16, with: 25 (7 PrSMs) Without: 16, with: 26 (3 PrSMs) Without: 16, with: 27 (3 PrSMs) Without: 16, with: 28 (1 PrSM) Without: 16, with: 29 (3 PrSMs) Without: 16, with: 30 (3 PrSMs) Without: 17, with: 17 (2 PrSMs) Without: 17, with: 18 (2 PrSMs) Without: 17, with: 19 (7 PrSMs) Without: 17, with: 20 (10 PrSMs) Without: 17, with: 21 (11 PrSMs) Without: 17, with: 22 (12 PrSMs) Without: 17, with: 23 (8 PrSMs) Without: 17, with: 24 (10 PrSMs) Without: 17, with: 25 (4 PrSMs) Without: 17, with: 26 (7 PrSMs) Without: 17, with: 27 (4 PrSMs) Without: 17, with: 28 (4 PrSMs) Without: 17, with: 29 (2 PrSMs) Without: 17, with: 31 (1 PrSM) Without: 17, with: 32 (1 PrSM) Without: 17, with: 34 (1 PrSM) Without: 18, with: 18 (1 PrSM) Without: 18, with: 19 (5 PrSMs) Without: 18, with: 20 (3 PrSMs) Without: 18, with: 21 (9 PrSMs) Without: 18, with: 22 (5 PrSMs) Without: 18, with: 23 (11 PrSMs) Without: 18, with: 24 (10 PrSMs) Without: 18, with: 25 (9 PrSMs) Without: 18, with: 26 (6 PrSMs) Without: 18, with: 27 (7 PrSMs) Without: 18, with: 28 (5 PrSMs) Without: 18, with: 29 (3 PrSMs) Without: 18, with: 30 (1 PrSM) Without: 18, with: 31 (3 PrSMs) Without: 18, with: 32 (2 PrSMs) Without: 18, with: 33 (1 PrSM) Without: 18, with: 36 (1 PrSM) Without: 19, with: 19 (2 PrSMs) Without: 19, with: 20 (3 PrSMs) Without: 19, with: 21 (9 PrSMs) Without: 19, with: 22 (7 PrSMs) Without: 19, with: 23 (5 PrSMs) Without: 19, with: 24 (10 PrSMs) Without: 19, with: 25 (11 PrSMs) Without: 19, with: 26 (10 PrSMs) Without: 19, with: 27 (3 PrSMs) Without: 19, with: 28 (4 PrSMs) Without: 19, with: 29 (4 PrSMs) Without: 19, with: 30 (5 PrSMs) Without: 19, with: 31 (4 PrSMs) Without: 19, with: 33 (1 PrSM) Without: 19, with: 37 (1 PrSM) Without: 20, with: 20 (1 PrSM) Without: 20, with: 21 (4 PrSMs) Without: 20, with: 22 (4 PrSMs) Without: 20, with: 23 (5 PrSMs) Without: 20, with: 24 (10 PrSMs) Without: 20, with: 25 (4 PrSMs) Without: 20, with: 26 (8 PrSMs) Without: 20, with: 27 (5 PrSMs) Without: 20, with: 28 (9 PrSMs) Without: 20, with: 29 (7 PrSMs) Without: 20, with: 30 (3 PrSMs) Without: 20, with: 31 (1 PrSM) Without: 20, with: 33 (1 PrSM) Without: 20, with: 35 (1 PrSM) Without: 20, with: 36 (1 PrSM) Without: 21, with: 23 (7 PrSMs) Without: 21, with: 24 (1 PrSM) Without: 21, with: 25 (4 PrSMs) Without: 21, with: 26 (7 PrSMs) Without: 21, with: 27 (17 PrSMs) Without: 21, with: 28 (9 PrSMs) Without: 21, with: 29 (8 PrSMs) Without: 21, with: 30 (5 PrSMs) Without: 21, with: 31 (8 PrSMs) Without: 21, with: 32 (6 PrSMs) Without: 21, with: 33 (1 PrSM) Without: 21, with: 34 (1 PrSM) Without: 21, with: 35 (1 PrSM) Without: 21, with: 36 (2 PrSMs) Without: 22, with: 23 (5 PrSMs) Without: 22, with: 24 (7 PrSMs) Without: 22, with: 25 (4 PrSMs) Without: 22, with: 26 (7 PrSMs) Without: 22, with: 27 (6 PrSMs) Without: 22, with: 28 (9 PrSMs) Without: 22, with: 29 (8 PrSMs) Without: 22, with: 30 (11 PrSMs) Without: 22, with: 31 (6 PrSMs) Without: 22, with: 32 (3 PrSMs) Without: 22, with: 33 (8 PrSMs) Without: 22, with: 34 (5 PrSMs) Without: 22, with: 37 (3 PrSMs) Without: 22, with: 38 (1 PrSM) Without: 22, with: 41 (1 PrSM) Without: 23, with: 24 (4 PrSMs) Without: 23, with: 25 (5 PrSMs) Without: 23, with: 26 (3 PrSMs) Without: 23, with: 27 (5 PrSMs) Without: 23, with: 28 (10 PrSMs) Without: 23, with: 29 (9 PrSMs) Without: 23, with: 30 (6 PrSMs) Without: 23, with: 31 (9 PrSMs) Without: 23, with: 32 (8 PrSMs) Without: 23, with: 33 (7 PrSMs) Without: 23, with: 34 (1 PrSM) Without: 23, with: 35 (2 PrSMs) Without: 23, with: 36 (1 PrSM) Without: 23, with: 37 (2 PrSMs) Without: 23, with: 39 (2 PrSMs) Without: 24, with: 26 (6 PrSMs) Without: 24, with: 27 (9 PrSMs) Without: 24, with: 28 (6 PrSMs) Without: 24, with: 29 (7 PrSMs) Without: 24, with: 30 (15 PrSMs) Without: 24, with: 31 (3 PrSMs) Without: 24, with: 32 (12 PrSMs) Without: 24, with: 33 (5 PrSMs) Without: 24, with: 34 (5 PrSMs) Without: 24, with: 35 (7 PrSMs) Without: 24, with: 36 (2 PrSMs) Without: 24, with: 37 (1 PrSM) Without: 25, with: 26 (1 PrSM) Without: 25, with: 27 (2 PrSMs) Without: 25, with: 28 (2 PrSMs) Without: 25, with: 29 (11 PrSMs) Without: 25, with: 30 (2 PrSMs) Without: 25, with: 31 (9 PrSMs) Without: 25, with: 32 (9 PrSMs) Without: 25, with: 33 (9 PrSMs) Without: 25, with: 34 (11 PrSMs) Without: 25, with: 35 (4 PrSMs) Without: 25, with: 36 (2 PrSMs) Without: 25, with: 37 (1 PrSM) Without: 25, with: 38 (2 PrSMs) Without: 25, with: 39 (1 PrSM) Without: 25, with: 40 (2 PrSMs) Without: 26, with: 26 (1 PrSM) Without: 26, with: 27 (1 PrSM) Without: 26, with: 28 (4 PrSMs) Without: 26, with: 29 (3 PrSMs) Without: 26, with: 30 (6 PrSMs) Without: 26, with: 31 (7 PrSMs) Without: 26, with: 32 (9 PrSMs) Without: 26, with: 33 (12 PrSMs) Without: 26, with: 34 (3 PrSMs) Without: 26, with: 35 (10 PrSMs) Without: 26, with: 36 (4 PrSMs) Without: 26, with: 37 (5 PrSMs) Without: 26, with: 38 (3 PrSMs) Without: 26, with: 39 (3 PrSMs) Without: 27, with: 28 (2 PrSMs) Without: 27, with: 29 (3 PrSMs) Without: 27, with: 30 (9 PrSMs) Without: 27, with: 31 (6 PrSMs) Without: 27, with: 32 (8 PrSMs) Without: 27, with: 33 (9 PrSMs) Without: 27, with: 34 (9 PrSMs) Without: 27, with: 35 (7 PrSMs) Without: 27, with: 36 (5 PrSMs) Without: 27, with: 37 (4 PrSMs) Without: 27, with: 38 (5 PrSMs) Without: 27, with: 39 (3 PrSMs) Without: 27, with: 40 (1 PrSM) Without: 27, with: 42 (2 PrSMs) Without: 27, with: 43 (3 PrSMs) Without: 28, with: 30 (2 PrSMs) Without: 28, with: 31 (5 PrSMs) Without: 28, with: 32 (12 PrSMs) Without: 28, with: 33 (8 PrSMs) Without: 28, with: 34 (6 PrSMs) Without: 28, with: 35 (6 PrSMs) Without: 28, with: 36 (4 PrSMs) Without: 28, with: 37 (6 PrSMs) Without: 28, with: 38 (3 PrSMs) Without: 28, with: 39 (5 PrSMs) Without: 28, with: 41 (4 PrSMs) Without: 28, with: 42 (1 PrSM) Without: 29, with: 29 (1 PrSM) Without: 29, with: 30 (3 PrSMs) Without: 29, with: 31 (3 PrSMs) Without: 29, with: 32 (2 PrSMs) Without: 29, with: 33 (7 PrSMs) Without: 29, with: 34 (10 PrSMs) Without: 29, with: 35 (8 PrSMs) Without: 29, with: 36 (10 PrSMs) Without: 29, with: 37 (4 PrSMs) Without: 29, with: 38 (7 PrSMs) Without: 29, with: 39 (3 PrSMs) Without: 29, with: 40 (7 PrSMs) Without: 29, with: 41 (4 PrSMs) Without: 29, with: 44 (1 PrSM) Without: 29, with: 45 (1 PrSM) Without: 30, with: 31 (1 PrSM) Without: 30, with: 32 (5 PrSMs) Without: 30, with: 33 (5 PrSMs) Without: 30, with: 34 (7 PrSMs) Without: 30, with: 35 (10 PrSMs) Without: 30, with: 36 (11 PrSMs) Without: 30, with: 37 (8 PrSMs) Without: 30, with: 38 (10 PrSMs) Without: 30, with: 39 (5 PrSMs) Without: 30, with: 40 (6 PrSMs) Without: 30, with: 41 (5 PrSMs) Without: 30, with: 42 (2 PrSMs) Without: 30, with: 43 (4 PrSMs) Without: 30, with: 45 (1 PrSM) Without: 31, with: 33 (3 PrSMs) Without: 31, with: 34 (2 PrSMs) Without: 31, with: 35 (7 PrSMs) Without: 31, with: 36 (12 PrSMs) Without: 31, with: 37 (9 PrSMs) Without: 31, with: 38 (5 PrSMs) Without: 31, with: 39 (5 PrSMs) Without: 31, with: 40 (7 PrSMs) Without: 31, with: 41 (4 PrSMs) Without: 31, with: 42 (2 PrSMs) Without: 31, with: 43 (5 PrSMs) Without: 31, with: 44 (1 PrSM) Without: 31, with: 45 (1 PrSM) Without: 32, with: 32 (1 PrSM) Without: 32, with: 34 (4 PrSMs) Without: 32, with: 35 (1 PrSM) Without: 32, with: 36 (7 PrSMs) Without: 32, with: 37 (4 PrSMs) Without: 32, with: 38 (4 PrSMs) Without: 32, with: 39 (13 PrSMs) Without: 32, with: 40 (7 PrSMs) Without: 32, with: 41 (9 PrSMs) Without: 32, with: 42 (2 PrSMs) Without: 32, with: 43 (1 PrSM) Without: 32, with: 44 (3 PrSMs) Without: 32, with: 45 (1 PrSM) Without: 32, with: 46 (2 PrSMs) Without: 33, with: 34 (1 PrSM) Without: 33, with: 35 (5 PrSMs) Without: 33, with: 36 (2 PrSMs) Without: 33, with: 37 (8 PrSMs) Without: 33, with: 38 (5 PrSMs) Without: 33, with: 39 (6 PrSMs) Without: 33, with: 40 (9 PrSMs) Without: 33, with: 41 (2 PrSMs) Without: 33, with: 42 (5 PrSMs) Without: 33, with: 43 (8 PrSMs) Without: 33, with: 44 (1 PrSM) Without: 33, with: 45 (2 PrSMs) Without: 33, with: 46 (1 PrSM) Without: 33, with: 48 (1 PrSM) Without: 34, with: 34 (2 PrSMs) Without: 34, with: 35 (1 PrSM) Without: 34, with: 36 (1 PrSM) Without: 34, with: 37 (5 PrSMs) Without: 34, with: 38 (5 PrSMs) Without: 34, with: 39 (5 PrSMs) Without: 34, with: 40 (3 PrSMs) Without: 34, with: 41 (7 PrSMs) Without: 34, with: 42 (7 PrSMs) Without: 34, with: 43 (9 PrSMs) Without: 34, with: 44 (1 PrSM) Without: 34, with: 45 (3 PrSMs) Without: 34, with: 46 (4 PrSMs) Without: 34, with: 47 (2 PrSMs) Without: 34, with: 48 (2 PrSMs) Without: 35, with: 37 (1 PrSM) Without: 35, with: 38 (3 PrSMs) Without: 35, with: 39 (6 PrSMs) Without: 35, with: 40 (7 PrSMs) Without: 35, with: 41 (4 PrSMs) Without: 35, with: 42 (4 PrSMs) Without: 35, with: 43 (7 PrSMs) Without: 35, with: 44 (6 PrSMs) Without: 35, with: 45 (3 PrSMs) Without: 35, with: 46 (4 PrSMs) Without: 35, with: 47 (1 PrSM) Without: 35, with: 48 (1 PrSM) Without: 36, with: 37 (1 PrSM) Without: 36, with: 38 (1 PrSM) Without: 36, with: 39 (4 PrSMs) Without: 36, with: 40 (9 PrSMs) Without: 36, with: 41 (5 PrSMs) Without: 36, with: 42 (8 PrSMs) Without: 36, with: 43 (9 PrSMs) Without: 36, with: 44 (4 PrSMs) Without: 36, with: 45 (5 PrSMs) Without: 36, with: 46 (4 PrSMs) Without: 36, with: 47 (3 PrSMs) Without: 36, with: 48 (2 PrSMs) Without: 36, with: 49 (3 PrSMs) Without: 37, with: 40 (5 PrSMs) Without: 37, with: 41 (4 PrSMs) Without: 37, with: 42 (8 PrSMs) Without: 37, with: 43 (8 PrSMs) Without: 37, with: 44 (6 PrSMs) Without: 37, with: 45 (8 PrSMs) Without: 37, with: 46 (5 PrSMs) Without: 37, with: 47 (4 PrSMs) Without: 37, with: 48 (5 PrSMs) Without: 37, with: 49 (1 PrSM) Without: 38, with: 39 (1 PrSM) Without: 38, with: 40 (2 PrSMs) Without: 38, with: 41 (4 PrSMs) Without: 38, with: 42 (5 PrSMs) Without: 38, with: 43 (10 PrSMs) Without: 38, with: 44 (8 PrSMs) Without: 38, with: 45 (8 PrSMs) Without: 38, with: 46 (10 PrSMs) Without: 38, with: 47 (5 PrSMs) Without: 38, with: 48 (3 PrSMs) Without: 38, with: 49 (4 PrSMs) Without: 38, with: 50 (1 PrSM) Without: 39, with: 42 (2 PrSMs) Without: 39, with: 43 (1 PrSM) Without: 39, with: 44 (8 PrSMs) Without: 39, with: 45 (3 PrSMs) Without: 39, with: 46 (4 PrSMs) Without: 39, with: 47 (7 PrSMs) Without: 39, with: 48 (7 PrSMs) Without: 39, with: 49 (7 PrSMs) Without: 39, with: 51 (1 PrSM) Without: 39, with: 52 (1 PrSM) Without: 39, with: 53 (1 PrSM) Without: 40, with: 41 (1 PrSM) Without: 40, with: 42 (4 PrSMs) Without: 40, with: 43 (3 PrSMs) Without: 40, with: 44 (5 PrSMs) Without: 40, with: 45 (8 PrSMs) Without: 40, with: 46 (5 PrSMs) Without: 40, with: 47 (7 PrSMs) Without: 40, with: 48 (4 PrSMs) Without: 40, with: 49 (4 PrSMs) Without: 40, with: 50 (6 PrSMs) Without: 40, with: 51 (5 PrSMs) Without: 40, with: 52 (2 PrSMs) Without: 40, with: 54 (1 PrSM) Without: 41, with: 42 (1 PrSM) Without: 41, with: 43 (2 PrSMs) Without: 41, with: 44 (2 PrSMs) Without: 41, with: 45 (5 PrSMs) Without: 41, with: 46 (3 PrSMs) Without: 41, with: 47 (11 PrSMs) Without: 41, with: 48 (8 PrSMs) Without: 41, with: 49 (3 PrSMs) Without: 41, with: 50 (8 PrSMs) Without: 41, with: 51 (2 PrSMs) Without: 41, with: 52 (3 PrSMs) Without: 41, with: 53 (1 PrSM) Without: 41, with: 54 (3 PrSMs) Without: 42, with: 44 (5 PrSMs) Without: 42, with: 45 (7 PrSMs) Without: 42, with: 46 (7 PrSMs) Without: 42, with: 47 (4 PrSMs) Without: 42, with: 48 (6 PrSMs) Without: 42, with: 49 (6 PrSMs) Without: 42, with: 50 (4 PrSMs) Without: 42, with: 51 (4 PrSMs) Without: 42, with: 52 (3 PrSMs) Without: 42, with: 53 (3 PrSMs) Without: 42, with: 54 (2 PrSMs) Without: 42, with: 56 (1 PrSM) Without: 43, with: 43 (1 PrSM) Without: 43, with: 45 (5 PrSMs) Without: 43, with: 46 (4 PrSMs) Without: 43, with: 47 (6 PrSMs) Without: 43, with: 48 (3 PrSMs) Without: 43, with: 49 (6 PrSMs) Without: 43, with: 50 (3 PrSMs) Without: 43, with: 51 (7 PrSMs) Without: 43, with: 52 (5 PrSMs) Without: 43, with: 53 (1 PrSM) Without: 43, with: 54 (2 PrSMs) Without: 43, with: 56 (2 PrSMs) Without: 43, with: 57 (1 PrSM) Without: 43, with: 62 (1 PrSM) Without: 44, with: 46 (2 PrSMs) Without: 44, with: 47 (2 PrSMs) Without: 44, with: 48 (5 PrSMs) Without: 44, with: 49 (4 PrSMs) Without: 44, with: 50 (4 PrSMs) Without: 44, with: 51 (5 PrSMs) Without: 44, with: 52 (1 PrSM) Without: 44, with: 53 (3 PrSMs) Without: 44, with: 54 (5 PrSMs) Without: 44, with: 55 (1 PrSM) Without: 44, with: 56 (3 PrSMs) Without: 44, with: 57 (1 PrSM) Without: 44, with: 60 (1 PrSM) Without: 45, with: 46 (1 PrSM) Without: 45, with: 47 (4 PrSMs) Without: 45, with: 48 (3 PrSMs) Without: 45, with: 49 (9 PrSMs) Without: 45, with: 50 (6 PrSMs) Without: 45, with: 51 (6 PrSMs) Without: 45, with: 52 (2 PrSMs) Without: 45, with: 53 (6 PrSMs) Without: 45, with: 54 (1 PrSM) Without: 45, with: 55 (3 PrSMs) Without: 45, with: 56 (4 PrSMs) Without: 45, with: 57 (1 PrSM) Without: 45, with: 60 (1 PrSM) Without: 46, with: 47 (3 PrSMs) Without: 46, with: 48 (1 PrSM) Without: 46, with: 49 (1 PrSM) Without: 46, with: 50 (10 PrSMs) Without: 46, with: 51 (4 PrSMs) Without: 46, with: 52 (3 PrSMs) Without: 46, with: 53 (3 PrSMs) Without: 46, with: 54 (8 PrSMs) Without: 46, with: 55 (6 PrSMs) Without: 46, with: 56 (1 PrSM) Without: 46, with: 57 (3 PrSMs) Without: 46, with: 58 (3 PrSMs) Without: 46, with: 59 (1 PrSM) Without: 46, with: 61 (2 PrSMs) Without: 47, with: 50 (2 PrSMs) Without: 47, with: 51 (3 PrSMs) Without: 47, with: 52 (5 PrSMs) Without: 47, with: 53 (2 PrSMs) Without: 47, with: 54 (4 PrSMs) Without: 47, with: 55 (2 PrSMs) Without: 47, with: 56 (6 PrSMs) Without: 47, with: 57 (3 PrSMs) Without: 47, with: 59 (1 PrSM) Without: 47, with: 64 (1 PrSM) Without: 48, with: 49 (1 PrSM) Without: 48, with: 50 (2 PrSMs) Without: 48, with: 51 (1 PrSM) Without: 48, with: 52 (2 PrSMs) Without: 48, with: 53 (4 PrSMs) Without: 48, with: 54 (5 PrSMs) Without: 48, with: 55 (7 PrSMs) Without: 48, with: 56 (3 PrSMs) Without: 48, with: 57 (3 PrSMs) Without: 48, with: 58 (1 PrSM) Without: 48, with: 59 (3 PrSMs) Without: 49, with: 49 (1 PrSM) Without: 49, with: 51 (3 PrSMs) Without: 49, with: 53 (2 PrSMs) Without: 49, with: 54 (7 PrSMs) Without: 49, with: 55 (4 PrSMs) Without: 49, with: 56 (9 PrSMs) Without: 49, with: 57 (2 PrSMs) Without: 49, with: 58 (4 PrSMs) Without: 49, with: 60 (1 PrSM) Without: 49, with: 61 (1 PrSM) Without: 50, with: 51 (2 PrSMs) Without: 50, with: 53 (4 PrSMs) Without: 50, with: 54 (2 PrSMs) Without: 50, with: 55 (2 PrSMs) Without: 50, with: 56 (2 PrSMs) Without: 50, with: 57 (1 PrSM) Without: 50, with: 58 (2 PrSMs) Without: 50, with: 59 (3 PrSMs) Without: 50, with: 60 (3 PrSMs) Without: 50, with: 61 (1 PrSM) Without: 50, with: 62 (3 PrSMs) Without: 50, with: 63 (1 PrSM) Without: 51, with: 54 (5 PrSMs) Without: 51, with: 55 (1 PrSM) Without: 51, with: 56 (5 PrSMs) Without: 51, with: 57 (7 PrSMs) Without: 51, with: 58 (4 PrSMs) Without: 51, with: 59 (4 PrSMs) Without: 51, with: 60 (1 PrSM) Without: 51, with: 61 (3 PrSMs) Without: 51, with: 62 (3 PrSMs) Without: 51, with: 63 (3 PrSMs) Without: 51, with: 65 (1 PrSM) Without: 52, with: 54 (2 PrSMs) Without: 52, with: 55 (3 PrSMs) Without: 52, with: 56 (1 PrSM) Without: 52, with: 57 (3 PrSMs) Without: 52, with: 58 (6 PrSMs) Without: 52, with: 59 (2 PrSMs) Without: 52, with: 60 (3 PrSMs) Without: 52, with: 61 (2 PrSMs) Without: 52, with: 62 (3 PrSMs) Without: 52, with: 63 (1 PrSM) Without: 52, with: 65 (1 PrSM) Without: 53, with: 53 (1 PrSM) Without: 53, with: 54 (1 PrSM) Without: 53, with: 56 (2 PrSMs) Without: 53, with: 57 (3 PrSMs) Without: 53, with: 58 (2 PrSMs) Without: 53, with: 59 (2 PrSMs) Without: 53, with: 60 (9 PrSMs) Without: 53, with: 61 (2 PrSMs) Without: 53, with: 62 (1 PrSM) Without: 53, with: 63 (3 PrSMs) Without: 53, with: 69 (1 PrSM) Without: 54, with: 57 (3 PrSMs) Without: 54, with: 58 (5 PrSMs) Without: 54, with: 59 (2 PrSMs) Without: 54, with: 60 (4 PrSMs) Without: 54, with: 61 (1 PrSM) Without: 54, with: 62 (2 PrSMs) Without: 54, with: 63 (1 PrSM) Without: 54, with: 64 (1 PrSM) Without: 54, with: 71 (1 PrSM) Without: 55, with: 56 (1 PrSM) Without: 55, with: 58 (1 PrSM) Without: 55, with: 59 (3 PrSMs) Without: 55, with: 60 (2 PrSMs) Without: 55, with: 61 (5 PrSMs) Without: 55, with: 62 (4 PrSMs) Without: 55, with: 64 (2 PrSMs) Without: 55, with: 65 (1 PrSM) Without: 55, with: 67 (2 PrSMs) Without: 55, with: 72 (1 PrSM) Without: 55, with: 73 (1 PrSM) Without: 56, with: 57 (2 PrSMs) Without: 56, with: 58 (2 PrSMs) Without: 56, with: 59 (1 PrSM) Without: 56, with: 60 (2 PrSMs) Without: 56, with: 62 (4 PrSMs) Without: 56, with: 63 (5 PrSMs) Without: 56, with: 65 (4 PrSMs) Without: 56, with: 68 (2 PrSMs) Without: 56, with: 69 (1 PrSM) Without: 57, with: 58 (1 PrSM) Without: 57, with: 59 (2 PrSMs) Without: 57, with: 60 (2 PrSMs) Without: 57, with: 61 (3 PrSMs) Without: 57, with: 62 (1 PrSM) Without: 57, with: 63 (3 PrSMs) Without: 57, with: 64 (3 PrSMs) Without: 57, with: 65 (6 PrSMs) Without: 57, with: 67 (1 PrSM) Without: 57, with: 72 (1 PrSM) Without: 57, with: 73 (1 PrSM) Without: 58, with: 60 (2 PrSMs) Without: 58, with: 61 (2 PrSMs) Without: 58, with: 63 (2 PrSMs) Without: 58, with: 64 (2 PrSMs) Without: 58, with: 65 (3 PrSMs) Without: 58, with: 66 (3 PrSMs) Without: 58, with: 68 (3 PrSMs) Without: 58, with: 70 (1 PrSM) Without: 58, with: 71 (1 PrSM) Without: 58, with: 72 (1 PrSM) Without: 59, with: 61 (2 PrSMs) Without: 59, with: 62 (1 PrSM) Without: 59, with: 63 (3 PrSMs) Without: 59, with: 64 (2 PrSMs) Without: 59, with: 65 (1 PrSM) Without: 59, with: 66 (2 PrSMs) Without: 59, with: 67 (2 PrSMs) Without: 59, with: 68 (1 PrSM) Without: 59, with: 69 (3 PrSMs) Without: 59, with: 70 (3 PrSMs) Without: 59, with: 71 (2 PrSMs) Without: 59, with: 74 (1 PrSM) Without: 60, with: 61 (2 PrSMs) Without: 60, with: 62 (2 PrSMs) Without: 60, with: 63 (4 PrSMs) Without: 60, with: 64 (1 PrSM) Without: 60, with: 65 (2 PrSMs) Without: 60, with: 66 (5 PrSMs) Without: 60, with: 67 (6 PrSMs) Without: 60, with: 68 (2 PrSMs) Without: 60, with: 69 (2 PrSMs) Without: 60, with: 70 (2 PrSMs) Without: 60, with: 71 (1 PrSM) Without: 61, with: 63 (2 PrSMs) Without: 61, with: 64 (5 PrSMs) Without: 61, with: 65 (1 PrSM) Without: 61, with: 66 (2 PrSMs) Without: 61, with: 67 (1 PrSM) Without: 61, with: 68 (1 PrSM) Without: 61, with: 69 (4 PrSMs) Without: 61, with: 70 (2 PrSMs) Without: 61, with: 71 (1 PrSM) Without: 62, with: 62 (1 PrSM) Without: 62, with: 64 (2 PrSMs) Without: 62, with: 66 (3 PrSMs) Without: 62, with: 68 (1 PrSM) Without: 62, with: 69 (1 PrSM) Without: 62, with: 70 (4 PrSMs) Without: 62, with: 71 (1 PrSM) Without: 62, with: 72 (1 PrSM) Without: 63, with: 64 (1 PrSM) Without: 63, with: 65 (2 PrSMs) Without: 63, with: 66 (1 PrSM) Without: 63, with: 67 (3 PrSMs) Without: 63, with: 68 (1 PrSM) Without: 63, with: 69 (2 PrSMs) Without: 63, with: 70 (1 PrSM) Without: 63, with: 72 (1 PrSM) Without: 63, with: 73 (3 PrSMs) Without: 64, with: 65 (1 PrSM) Without: 64, with: 68 (2 PrSMs) Without: 64, with: 69 (3 PrSMs) Without: 64, with: 70 (1 PrSM) Without: 64, with: 72 (1 PrSM) Without: 64, with: 73 (1 PrSM) Without: 64, with: 74 (3 PrSMs) Without: 64, with: 75 (1 PrSM) Without: 64, with: 76 (2 PrSMs) Without: 65, with: 65 (1 PrSM) Without: 65, with: 68 (1 PrSM) Without: 65, with: 69 (1 PrSM) Without: 65, with: 70 (2 PrSMs) Without: 65, with: 71 (2 PrSMs) Without: 65, with: 72 (1 PrSM) Without: 65, with: 73 (1 PrSM) Without: 65, with: 75 (1 PrSM) Without: 65, with: 76 (1 PrSM) Without: 65, with: 86 (1 PrSM) Without: 66, with: 68 (1 PrSM) Without: 66, with: 69 (1 PrSM) Without: 66, with: 70 (3 PrSMs) Without: 66, with: 72 (1 PrSM) Without: 66, with: 73 (2 PrSMs) Without: 66, with: 74 (1 PrSM) Without: 66, with: 76 (1 PrSM) Without: 66, with: 78 (1 PrSM) Without: 66, with: 84 (1 PrSM) Without: 67, with: 69 (2 PrSMs) Without: 67, with: 70 (2 PrSMs) Without: 67, with: 71 (1 PrSM) Without: 67, with: 72 (3 PrSMs) Without: 67, with: 73 (1 PrSM) Without: 67, with: 75 (2 PrSMs) Without: 67, with: 77 (1 PrSM) Without: 68, with: 70 (1 PrSM) Without: 68, with: 71 (1 PrSM) Without: 68, with: 72 (3 PrSMs) Without: 68, with: 73 (1 PrSM) Without: 68, with: 74 (2 PrSMs) Without: 68, with: 75 (3 PrSMs) Without: 68, with: 77 (1 PrSM) Without: 68, with: 78 (1 PrSM) Without: 68, with: 79 (1 PrSM) Without: 69, with: 70 (1 PrSM) Without: 69, with: 71 (1 PrSM) Without: 69, with: 72 (2 PrSMs) Without: 69, with: 73 (1 PrSM) Without: 69, with: 74 (1 PrSM) Without: 69, with: 75 (1 PrSM) Without: 69, with: 77 (2 PrSMs) Without: 69, with: 79 (1 PrSM) Without: 70, with: 71 (1 PrSM) Without: 70, with: 72 (1 PrSM) Without: 70, with: 74 (3 PrSMs) Without: 70, with: 75 (2 PrSMs) Without: 70, with: 76 (2 PrSMs) Without: 70, with: 77 (1 PrSM) Without: 70, with: 79 (2 PrSMs) Without: 70, with: 80 (2 PrSMs) Without: 70, with: 81 (1 PrSM) Without: 71, with: 73 (1 PrSM) Without: 71, with: 75 (2 PrSMs) Without: 71, with: 76 (2 PrSMs) Without: 71, with: 77 (1 PrSM) Without: 71, with: 79 (4 PrSMs) Without: 71, with: 80 (2 PrSMs) Without: 71, with: 82 (2 PrSMs) Without: 72, with: 74 (3 PrSMs) Without: 72, with: 75 (2 PrSMs) Without: 72, with: 76 (1 PrSM) Without: 72, with: 77 (2 PrSMs) Without: 72, with: 79 (1 PrSM) Without: 72, with: 80 (1 PrSM) Without: 72, with: 83 (1 PrSM) Without: 73, with: 74 (1 PrSM) Without: 73, with: 76 (1 PrSM) Without: 73, with: 77 (1 PrSM) Without: 73, with: 78 (1 PrSM) Without: 73, with: 79 (1 PrSM) Without: 73, with: 80 (1 PrSM) Without: 73, with: 82 (1 PrSM) Without: 73, with: 83 (1 PrSM) Without: 74, with: 77 (1 PrSM) Without: 74, with: 78 (1 PrSM) Without: 74, with: 79 (2 PrSMs) Without: 75, with: 78 (1 PrSM) Without: 75, with: 79 (1 PrSM) Without: 75, with: 80 (1 PrSM) Without: 75, with: 81 (2 PrSMs) Without: 75, with: 82 (1 PrSM) Without: 76, with: 78 (1 PrSM) Without: 76, with: 79 (3 PrSMs) Without: 76, with: 81 (1 PrSM) Without: 76, with: 82 (1 PrSM) Without: 76, with: 86 (2 PrSMs) Without: 77, with: 80 (1 PrSM) Without: 77, with: 82 (1 PrSM) Without: 77, with: 87 (1 PrSM) Without: 78, with: 82 (3 PrSMs) Without: 78, with: 83 (1 PrSM) Without: 78, with: 85 (1 PrSM) Without: 78, with: 86 (1 PrSM) Without: 79, with: 83 (1 PrSM) Without: 79, with: 85 (1 PrSM) Without: 79, with: 90 (1 PrSM) Without: 80, with: 80 (1 PrSM) Without: 80, with: 83 (1 PrSM) Without: 80, with: 84 (1 PrSM) Without: 80, with: 86 (1 PrSM) Without: 80, with: 90 (1 PrSM) Without: 80, with: 91 (1 PrSM) Without: 81, with: 86 (1 PrSM) Without: 81, with: 88 (1 PrSM) Without: 81, with: 94 (1 PrSM) Without: 81, with: 95 (1 PrSM) Without: 82, with: 87 (2 PrSMs) Without: 82, with: 89 (1 PrSM) Without: 82, with: 94 (1 PrSM) Without: 83, with: 87 (1 PrSM) Without: 83, with: 91 (1 PrSM) Without: 84, with: 87 (1 PrSM) Without: 84, with: 91 (1 PrSM) Without: 85, with: 86 (1 PrSM) Without: 85, with: 87 (1 PrSM) Without: 85, with: 96 (1 PrSM) Without: 86, with: 89 (1 PrSM) Without: 86, with: 90 (2 PrSMs) Without: 86, with: 91 (3 PrSMs) Without: 86, with: 93 (2 PrSMs) Without: 87, with: 89 (1 PrSM) Without: 87, with: 92 (1 PrSM) Without: 87, with: 94 (1 PrSM) Without: 88, with: 90 (1 PrSM) Without: 88, with: 92 (1 PrSM) Without: 88, with: 94 (1 PrSM) Without: 88, with: 95 (1 PrSM) Without: 88, with: 100 (1 PrSM) Without: 89, with: 95 (1 PrSM) Without: 89, with: 103 (1 PrSM) Without: 90, with: 94 (1 PrSM) Without: 91, with: 95 (1 PrSM) Without: 91, with: 98 (1 PrSM) Without: 91, with: 101 (1 PrSM) Without: 92, with: 97 (2 PrSMs) Without: 92, with: 98 (1 PrSM) Without: 92, with: 101 (1 PrSM) Without: 92, with: 102 (1 PrSM) Without: 93, with: 99 (1 PrSM) Without: 94, with: 99 (1 PrSM) Without: 94, with: 103 (1 PrSM) Without: 95, with: 104 (1 PrSM) Without: 96, with: 100 (1 PrSM) Without: 96, with: 106 (2 PrSMs) Without: 97, with: 102 (1 PrSM) Without: 98, with: 103 (1 PrSM) Without: 99, with: 106 (1 PrSM) Without: 100, with: 106 (1 PrSM) Without: 101, with: 106 (1 PrSM) Without: 107, with: 107 (1 PrSM) Without: 107, with: 111 (1 PrSM) Without: 108, with: 114 (1 PrSM) Without: 111, with: 118 (1 PrSM) Without: 112, with: 120 (1 PrSM) Without: 113, with: 118 (1 PrSM) Without: 113, with: 124 (1 PrSM) Without: 121, with: 128 (1 PrSM) Without: 122, with: 130 (1 PrSM) Without post mass matching With post mass matching

Each dot is one PrSM identified by both runs; its position gives the number of matched fragment masses without (x) and with (y) post mass matching. Dots above the diagonal line gained matched masses.

Per PrSM (median)Without post mass matchingWith post mass matching
Masses in the spectrum170177
Matched fragment masses2936
Matched fragment ions2229

Post mass matching added a median of 6 matched fragment masses per PrSM (interquartile range 4 to 9, maximum 21). The number of matched fragment ions per PrSM rose from a median of 22 to 29. The added masses are mostly small (median 0.85 kDa) and carry low charges (86% are singly or doubly charged).

The two runs report the same 3,207 PrSMs, 1,918 proteoforms, and 436 proteins with the same E-values: post mass matching changes the matched fragment masses of the PrSMs, not which spectra, proteoforms, and proteins are identified. Post mass matching takes 12 seconds of the 5.3-minute search.