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 2.0.0; 3.5 minutes, 7.3 GB of memory). The index takes 172 GB of disk space. TopPIC 1.8.1 and 1.9.0 used an index generated with the same options in their earlier file layout (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 1.8.1 vs 1.9.0 vs 2.0.0

This benchmark compares the identification results and the running times of TopFD and TopPIC in TopPIC Suite versions 1.8.1, 1.9.0, and 2.0.0 with their default settings: at most one unexpected mass shift and no variable modifications. All runs used 16 threads.

Settings

Version 1.8.1Version 1.9.0Version 2.0.0
TopFD commandtopfd -u 16 -g 480_F3_02.mzMLtopfd -u 16 480_F3_02.mzMLtopfd -u 16 480_F3_02.mzML
TopPIC commandtoppic -u 16 -f C57 -g -d -t FDR -T 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

The option -g (skip HTML output) exists only in version 1.8.1, which generates HTML reports by default; versions 1.9.0 and 2.0.0 no longer generate them. Versions 1.9.0 and 2.0.0 write an SQLite file with the centroided spectra in TopFD, which TopPIC uses for post mass matching (benchmark 4) and in which it stores the identifications; both steps are included in the running times. The protein-level cutoff (-y) is not available in version 1.8.1.

Results

Identifications at 1% FDR Identifications at 1% FDR Version 1.8.1 Version 1.9.0 Version 2.0.0 0 1,000 2,000 3,000 4,000 Number of identifications PrSMs, Version 1.8.1: 2,795 2,795 PrSMs, Version 1.9.0: 2,870 2,870 PrSMs, Version 2.0.0: 2,966 2,966 PrSMs Proteoforms, Version 1.8.1: 1,654 1,654 Proteoforms, Version 1.9.0: 1,686 1,686 Proteoforms, Version 2.0.0: 1,765 1,765 Proteoforms
Running time of TopFD Running time of TopFD 0 5 10 15 Wall-clock time (minutes) Version 1.8.1, TopFD: 12.6 minutes 12.6 Version 1.8.1 Version 1.9.0, TopFD: 12.6 minutes 12.6 Version 1.9.0 Version 2.0.0, TopFD: 5.7 minutes 5.7 Version 2.0.0
Running time of TopPIC Running time of TopPIC 0 20 40 60 80 Wall-clock time (minutes) Version 1.8.1, TopPIC: 48.4 minutes 48.4 Version 1.8.1 Version 1.9.0, TopPIC: 57.4 minutes 57.4 Version 1.9.0 Version 2.0.0, TopPIC: 6.2 minutes 6.2 Version 2.0.0
Version 1.8.1Version 1.9.0Version 2.0.0
PrSMs (1% spectrum-level FDR)2,7952,8702,966 (+3.3%)
Proteoforms (1% proteoform-level FDR)1,6541,6861,765 (+4.7%)
TopFD running time (minutes)12.612.65.7 (2.2X speedup)
TopPIC running time (minutes)48.457.46.2 (9.2X speedup)

Changes and speedups in parentheses are relative to version 1.9.0.

Version 2.0.0 identified 6.1% more PrSMs and 6.7% more proteoforms than version 1.8.1, and 3.3% more PrSMs and 4.7% more proteoforms than version 1.9.0. TopPIC 2.0.0 ran 7.8 times faster than version 1.8.1 and 9.2 times faster than version 1.9.0, mainly because its filtering step for proteoforms with one unexpected mass shift takes 2.2 minutes instead of 35 to 37 minutes. TopFD 2.0.0 ran 2.2 times faster than the earlier versions, mainly because its MS1 feature detection scores the candidate features in batches.

3 TopPIC 2.0.0 search settings

This benchmark compares four search settings of TopPIC 2.0.0 on the same data set, database, and computer as benchmark 2: the default setting (at most one unexpected mass shift and no variable modifications), a search with methionine oxidation as a variable modification (-b var_mods.txt), a search with up to two unexpected mass shifts (-s 2), and a search with both options. All four searches used the same TopFD 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-s 2At most 2 unexpected mass shifts
d-s 2 -b var_mods.txtAt most 2 unexpected mass shifts, variable oxidation on M

The file var_mods.txt defines a single variable modification: oxidation (+15.994915 Da) on methionine (Unimod 35).

Results

Running time of TopPIC Running time of TopPIC 0 5 10 15 20 Wall-clock time (minutes) a: default, TopPIC: 6.2 minutes 6.2 a: default b: -b, TopPIC: 7.8 minutes 7.8 b: -b c: -s 2, TopPIC: 14.9 minutes 14.9 c: -s 2 d: -s 2 -b, TopPIC: 16.7 minutes 16.7 d: -s 2 -b
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: 2,966 (68.6% of the 4,323 MS/MS spectra) 2,966 a: default, Proteoforms: 1,765 1,765 a: default b: -b, PrSMs: 3,028 (70.0% of the 4,323 MS/MS spectra) 3,028 b: -b, Proteoforms: 1,808 1,808 b: -b c: -s 2, PrSMs: 3,086 (71.4% of the 4,323 MS/MS spectra) 3,086 c: -s 2, Proteoforms: 1,838 1,838 c: -s 2 d: -s 2 -b, PrSMs: 3,141 (72.7% of the 4,323 MS/MS spectra) 3,141 d: -s 2 -b, Proteoforms: 1,870 1,870 d: -s 2 -b
SettingRunning time (minutes)Peak memoryPrSMsProteoformsProteins
a: default6.216.8 GB2,9661,765416
b: -b var_mods.txt7.817.8 GB3,028 (+2.1%)1,808 (+2.4%)418
c: -s 214.917.2 GB3,086 (+4.0%)1,838 (+4.1%)418
d: -s 2 -b var_mods.txt16.717.5 GB3,141 (+5.9%)1,870 (+5.9%)420

Running time

Variable oxidation (-b) adds about 1.5 minutes and at most 1 GB of 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 2.4 times, from 6.2 to 14.9 minutes. With both options, the search takes 16.7 minutes.

Identifications

Settings b, c, and d keep nearly all PrSMs of the default search: they lose 3, 7, and 10 of its 2,966 PrSMs, all with one unexpected mass shift and E-values near the cutoff. Setting b identifies 65 new spectra and adds 43 proteoforms, and it also re-explains 320 PrSMs that carried an unexpected mass shift in the default search as proteoforms with one to three methionine oxidations and no unexpected shift, with much better E-values (a median of 1,900 times lower). Setting c identifies 127 spectra that the default search does not, 122 of them as proteoforms with two unknown alterations that the one-shift search cannot explain, and adds 73 proteoforms. The two options are nearly independent: setting d identifies 185 new spectra, close to the 65 + 127 of settings b and c, and it gives the largest set: 3,141 PrSMs and 1,870 proteoforms, 5.9% more of each than the default setting. Protein counts change by at most 4, so both options refine how proteins are explained rather than which proteins are identified.

Recommendation: use -b var_mods.txt with methionine oxidation routinely, because it costs about 1.5 minutes and gives more accurate proteoform assignments. Add -s 2 when more than doubling the running time is acceptable.

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 output with the default search settings of TopPIC 2.0.0; the run with post mass matching is setting a of benchmark 3. Post mass matching reads the centroided peaks from the SQLite file that TopFD writes unless it is run with -N; without that file, TopPIC stops with an error unless post mass matching is turned off.

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: 2 PrSMs 0–4 5–9 matched masses, without post mass matching: 209 PrSMs 5–9 matched masses, with post mass matching: 32 PrSMs 5–9 10–14 matched masses, without post mass matching: 405 PrSMs 10–14 matched masses, with post mass matching: 137 PrSMs 10–14 15–19 matched masses, without post mass matching: 469 PrSMs 15–19 matched masses, with post mass matching: 261 PrSMs 15–19 20–24 matched masses, without post mass matching: 400 PrSMs 20–24 matched masses, with post mass matching: 340 PrSMs 20–24 25–29 matched masses, without post mass matching: 362 PrSMs 25–29 matched masses, with post mass matching: 339 PrSMs 25–29 30–34 matched masses, without post mass matching: 322 PrSMs 30–34 matched masses, with post mass matching: 373 PrSMs 30–34 35–39 matched masses, without post mass matching: 266 PrSMs 35–39 matched masses, with post mass matching: 338 PrSMs 35–39 40–44 matched masses, without post mass matching: 167 PrSMs 40–44 matched masses, with post mass matching: 292 PrSMs 40–44 45–49 matched masses, without post mass matching: 129 PrSMs 45–49 matched masses, with post mass matching: 244 PrSMs 45–49 50–54 matched masses, without post mass matching: 88 PrSMs 50–54 matched masses, with post mass matching: 198 PrSMs 50–54 55–59 matched masses, without post mass matching: 61 PrSMs 55–59 matched masses, with post mass matching: 132 PrSMs 55–59 60–64 matched masses, without post mass matching: 38 PrSMs 60–64 matched masses, with post mass matching: 97 PrSMs 60–64 65–69 matched masses, without post mass matching: 19 PrSMs 65–69 matched masses, with post mass matching: 75 PrSMs 65–69 70–74 matched masses, without post mass matching: 9 PrSMs 70–74 matched masses, with post mass matching: 40 PrSMs 70–74 75–79 matched masses, without post mass matching: 11 PrSMs 75–79 matched masses, with post mass matching: 25 PrSMs 75–79 80–84 matched masses, without post mass matching: 2 PrSMs 80–84 matched masses, with post mass matching: 18 PrSMs 80–84 85–89 matched masses, without post mass matching: 4 PrSMs 85–89 matched masses, with post mass matching: 11 PrSMs 85–89 90–94 matched masses, without post mass matching: 1 PrSMs 90–94 matched masses, with post mass matching: 5 PrSMs 90–94 95–99 matched masses, with post mass matching: 2 PrSMs 95–99 100–104 matched masses, with post mass matching: 2 PrSMs 100–104 105–109 matched masses, with post mass matching: 3 PrSMs 105–109 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 y = x Without: 4, with: 4 (2 PrSMs) Without: 4, with: 8 (1 PrSM) Without: 4, with: 11 (1 PrSM) Without: 5, with: 5 (4 PrSMs) Without: 5, with: 6 (5 PrSMs) Without: 5, with: 7 (1 PrSM) Without: 5, with: 8 (2 PrSMs) Without: 5, with: 9 (1 PrSM) Without: 5, with: 10 (1 PrSM) Without: 5, with: 13 (1 PrSM) Without: 5, with: 14 (1 PrSM) Without: 5, with: 20 (1 PrSM) Without: 6, with: 6 (3 PrSMs) Without: 6, with: 7 (4 PrSMs) Without: 6, with: 8 (2 PrSMs) Without: 6, with: 9 (2 PrSMs) Without: 6, with: 10 (3 PrSMs) Without: 6, with: 11 (3 PrSMs) Without: 6, with: 12 (1 PrSM) Without: 6, with: 13 (1 PrSM) Without: 6, with: 14 (2 PrSMs) Without: 6, with: 15 (1 PrSM) Without: 6, with: 16 (1 PrSM) Without: 6, with: 17 (1 PrSM) Without: 6, with: 18 (2 PrSMs) Without: 6, with: 20 (2 PrSMs) Without: 7, with: 7 (1 PrSM) Without: 7, with: 8 (2 PrSMs) Without: 7, with: 9 (1 PrSM) Without: 7, with: 10 (6 PrSMs) Without: 7, with: 11 (5 PrSMs) Without: 7, with: 12 (2 PrSMs) Without: 7, with: 13 (3 PrSMs) Without: 7, with: 14 (6 PrSMs) Without: 7, with: 15 (6 PrSMs) Without: 7, with: 16 (2 PrSMs) Without: 7, with: 17 (4 PrSMs) Without: 7, with: 18 (1 PrSM) Without: 7, with: 20 (1 PrSM) Without: 7, with: 21 (2 PrSMs) Without: 7, with: 22 (1 PrSM) Without: 7, with: 23 (2 PrSMs) Without: 8, with: 9 (1 PrSM) Without: 8, with: 10 (2 PrSMs) Without: 8, with: 11 (11 PrSMs) Without: 8, with: 12 (6 PrSMs) Without: 8, with: 13 (4 PrSMs) Without: 8, with: 14 (2 PrSMs) Without: 8, with: 15 (4 PrSMs) Without: 8, with: 16 (5 PrSMs) Without: 8, with: 17 (3 PrSMs) Without: 8, with: 18 (3 PrSMs) Without: 8, with: 19 (2 PrSMs) Without: 8, with: 20 (4 PrSMs) Without: 8, with: 21 (1 PrSM) Without: 9, with: 9 (2 PrSMs) Without: 9, with: 10 (6 PrSMs) Without: 9, with: 11 (6 PrSMs) Without: 9, with: 12 (9 PrSMs) Without: 9, with: 13 (5 PrSMs) Without: 9, with: 14 (5 PrSMs) Without: 9, with: 15 (5 PrSMs) Without: 9, with: 16 (4 PrSMs) Without: 9, with: 17 (3 PrSMs) Without: 9, with: 18 (7 PrSMs) Without: 9, with: 19 (5 PrSMs) Without: 9, with: 20 (4 PrSMs) Without: 9, with: 21 (4 PrSMs) Without: 9, with: 22 (1 PrSM) Without: 9, with: 23 (1 PrSM) Without: 9, with: 24 (3 PrSMs) Without: 9, with: 25 (1 PrSM) Without: 10, with: 11 (1 PrSM) Without: 10, with: 12 (4 PrSMs) Without: 10, with: 13 (8 PrSMs) Without: 10, with: 14 (6 PrSMs) Without: 10, with: 15 (8 PrSMs) Without: 10, with: 16 (2 PrSMs) Without: 10, with: 17 (4 PrSMs) Without: 10, with: 18 (5 PrSMs) Without: 10, with: 19 (9 PrSMs) Without: 10, with: 20 (6 PrSMs) Without: 10, with: 21 (4 PrSMs) Without: 10, with: 22 (2 PrSMs) Without: 10, with: 23 (2 PrSMs) Without: 10, with: 24 (1 PrSM) Without: 10, with: 26 (1 PrSM) Without: 11, with: 11 (1 PrSM) Without: 11, with: 12 (4 PrSMs) Without: 11, with: 13 (6 PrSMs) Without: 11, with: 14 (6 PrSMs) Without: 11, with: 15 (8 PrSMs) Without: 11, with: 16 (7 PrSMs) Without: 11, with: 17 (5 PrSMs) Without: 11, with: 18 (8 PrSMs) Without: 11, with: 19 (8 PrSMs) Without: 11, with: 20 (7 PrSMs) Without: 11, with: 21 (6 PrSMs) Without: 11, with: 23 (2 PrSMs) Without: 11, with: 24 (6 PrSMs) Without: 11, with: 25 (2 PrSMs) Without: 11, with: 26 (2 PrSMs) Without: 11, with: 27 (1 PrSM) Without: 11, with: 29 (2 PrSMs) Without: 11, with: 30 (1 PrSM) Without: 12, with: 12 (2 PrSMs) Without: 12, with: 13 (1 PrSM) Without: 12, with: 14 (5 PrSMs) Without: 12, with: 15 (4 PrSMs) Without: 12, with: 16 (4 PrSMs) Without: 12, with: 17 (16 PrSMs) Without: 12, with: 18 (5 PrSMs) Without: 12, with: 19 (9 PrSMs) Without: 12, with: 20 (3 PrSMs) Without: 12, with: 21 (8 PrSMs) Without: 12, with: 22 (10 PrSMs) Without: 12, with: 23 (5 PrSMs) Without: 12, with: 24 (5 PrSMs) Without: 12, with: 25 (6 PrSMs) Without: 12, with: 28 (1 PrSM) Without: 12, with: 29 (1 PrSM) Without: 12, with: 32 (1 PrSM) Without: 13, with: 14 (1 PrSM) Without: 13, with: 15 (5 PrSMs) Without: 13, with: 16 (5 PrSMs) Without: 13, with: 17 (11 PrSMs) Without: 13, with: 18 (8 PrSMs) Without: 13, with: 19 (9 PrSMs) Without: 13, with: 20 (7 PrSMs) Without: 13, with: 21 (5 PrSMs) Without: 13, with: 22 (10 PrSMs) Without: 13, with: 23 (4 PrSMs) Without: 13, with: 24 (6 PrSMs) Without: 13, with: 25 (9 PrSMs) Without: 13, with: 26 (4 PrSMs) Without: 13, with: 27 (1 PrSM) Without: 13, with: 29 (1 PrSM) Without: 13, with: 30 (2 PrSMs) Without: 13, with: 31 (2 PrSMs) Without: 13, with: 33 (1 PrSM) Without: 13, with: 34 (1 PrSM) Without: 14, with: 15 (2 PrSMs) Without: 14, with: 16 (2 PrSMs) Without: 14, with: 17 (4 PrSMs) Without: 14, with: 18 (5 PrSMs) Without: 14, with: 19 (7 PrSMs) Without: 14, with: 20 (9 PrSMs) Without: 14, with: 21 (6 PrSMs) Without: 14, with: 22 (9 PrSMs) Without: 14, with: 23 (5 PrSMs) Without: 14, with: 24 (7 PrSMs) Without: 14, with: 25 (5 PrSMs) Without: 14, with: 26 (5 PrSMs) Without: 14, with: 27 (3 PrSMs) Without: 14, with: 28 (4 PrSMs) Without: 14, with: 30 (2 PrSMs) Without: 14, with: 31 (4 PrSMs) Without: 14, with: 32 (1 PrSM) Without: 14, with: 34 (2 PrSMs) Without: 15, with: 16 (4 PrSMs) Without: 15, with: 17 (4 PrSMs) Without: 15, with: 18 (3 PrSMs) Without: 15, with: 19 (3 PrSMs) Without: 15, with: 20 (8 PrSMs) Without: 15, with: 21 (8 PrSMs) Without: 15, with: 22 (10 PrSMs) Without: 15, with: 23 (4 PrSMs) Without: 15, with: 24 (6 PrSMs) Without: 15, with: 25 (5 PrSMs) Without: 15, with: 26 (4 PrSMs) Without: 15, with: 27 (5 PrSMs) Without: 15, with: 28 (1 PrSM) Without: 15, with: 29 (2 PrSMs) Without: 15, with: 30 (2 PrSMs) Without: 15, with: 31 (1 PrSM) Without: 15, with: 32 (1 PrSM) Without: 15, with: 33 (1 PrSM) Without: 16, with: 16 (2 PrSMs) Without: 16, with: 17 (2 PrSMs) Without: 16, with: 18 (3 PrSMs) Without: 16, with: 19 (4 PrSMs) Without: 16, with: 20 (10 PrSMs) Without: 16, with: 21 (5 PrSMs) Without: 16, with: 22 (8 PrSMs) Without: 16, with: 23 (11 PrSMs) Without: 16, with: 24 (5 PrSMs) Without: 16, with: 25 (11 PrSMs) Without: 16, with: 26 (5 PrSMs) Without: 16, with: 27 (6 PrSMs) Without: 16, with: 28 (4 PrSMs) Without: 16, with: 29 (8 PrSMs) Without: 16, with: 30 (4 PrSMs) Without: 16, with: 31 (4 PrSMs) Without: 16, with: 32 (5 PrSMs) Without: 16, with: 33 (3 PrSMs) Without: 16, with: 34 (1 PrSM) Without: 16, with: 35 (2 PrSMs) Without: 16, with: 38 (1 PrSM) Without: 17, with: 18 (4 PrSMs) Without: 17, with: 19 (5 PrSMs) Without: 17, with: 20 (6 PrSMs) Without: 17, with: 21 (7 PrSMs) Without: 17, with: 22 (7 PrSMs) Without: 17, with: 23 (7 PrSMs) Without: 17, with: 24 (6 PrSMs) Without: 17, with: 25 (3 PrSMs) Without: 17, with: 26 (8 PrSMs) Without: 17, with: 27 (5 PrSMs) Without: 17, with: 28 (7 PrSMs) Without: 17, with: 29 (5 PrSMs) Without: 17, with: 30 (3 PrSMs) Without: 17, with: 31 (4 PrSMs) Without: 17, with: 32 (5 PrSMs) Without: 17, with: 33 (2 PrSMs) Without: 17, with: 35 (1 PrSM) Without: 17, with: 37 (1 PrSM) Without: 18, with: 18 (1 PrSM) Without: 18, with: 19 (7 PrSMs) Without: 18, with: 20 (3 PrSMs) Without: 18, with: 21 (7 PrSMs) Without: 18, with: 22 (3 PrSMs) Without: 18, with: 23 (12 PrSMs) Without: 18, with: 24 (7 PrSMs) Without: 18, with: 25 (16 PrSMs) Without: 18, with: 26 (13 PrSMs) Without: 18, with: 27 (5 PrSMs) Without: 18, with: 28 (7 PrSMs) Without: 18, with: 29 (7 PrSMs) Without: 18, with: 30 (3 PrSMs) Without: 18, with: 31 (7 PrSMs) Without: 18, with: 32 (3 PrSMs) Without: 18, with: 33 (1 PrSM) Without: 18, with: 34 (2 PrSMs) Without: 18, with: 35 (2 PrSMs) Without: 18, with: 36 (3 PrSMs) Without: 18, with: 37 (1 PrSM) Without: 18, with: 38 (2 PrSMs) Without: 19, with: 20 (4 PrSMs) Without: 19, with: 21 (2 PrSMs) Without: 19, with: 22 (4 PrSMs) Without: 19, with: 23 (2 PrSMs) Without: 19, with: 24 (4 PrSMs) Without: 19, with: 25 (5 PrSMs) Without: 19, with: 26 (5 PrSMs) Without: 19, with: 27 (11 PrSMs) Without: 19, with: 28 (13 PrSMs) Without: 19, with: 29 (8 PrSMs) Without: 19, with: 30 (11 PrSMs) Without: 19, with: 31 (10 PrSMs) Without: 19, with: 32 (3 PrSMs) Without: 19, with: 33 (2 PrSMs) Without: 19, with: 34 (5 PrSMs) Without: 19, with: 35 (1 PrSM) Without: 19, with: 36 (2 PrSMs) Without: 19, with: 38 (2 PrSMs) Without: 19, with: 41 (1 PrSM) Without: 20, with: 21 (2 PrSMs) Without: 20, with: 23 (7 PrSMs) Without: 20, with: 24 (2 PrSMs) Without: 20, with: 25 (5 PrSMs) Without: 20, with: 26 (2 PrSMs) Without: 20, with: 27 (14 PrSMs) Without: 20, with: 28 (6 PrSMs) Without: 20, with: 29 (5 PrSMs) Without: 20, with: 30 (4 PrSMs) Without: 20, with: 31 (7 PrSMs) Without: 20, with: 32 (5 PrSMs) Without: 20, with: 33 (5 PrSMs) Without: 20, with: 34 (1 PrSM) Without: 20, with: 35 (3 PrSMs) Without: 20, with: 36 (3 PrSMs) Without: 20, with: 37 (3 PrSMs) Without: 20, with: 38 (1 PrSM) Without: 20, with: 39 (1 PrSM) Without: 21, with: 22 (1 PrSM) Without: 21, with: 23 (1 PrSM) Without: 21, with: 24 (5 PrSMs) Without: 21, with: 25 (4 PrSMs) Without: 21, with: 26 (5 PrSMs) Without: 21, with: 27 (6 PrSMs) Without: 21, with: 28 (7 PrSMs) Without: 21, with: 29 (5 PrSMs) Without: 21, with: 30 (3 PrSMs) Without: 21, with: 31 (14 PrSMs) Without: 21, with: 32 (10 PrSMs) Without: 21, with: 33 (8 PrSMs) Without: 21, with: 34 (5 PrSMs) Without: 21, with: 35 (5 PrSMs) Without: 21, with: 36 (4 PrSMs) Without: 21, with: 37 (3 PrSMs) Without: 21, with: 38 (5 PrSMs) Without: 21, with: 39 (1 PrSM) Without: 21, with: 40 (1 PrSM) Without: 21, with: 41 (1 PrSM) Without: 22, with: 23 (2 PrSMs) Without: 22, with: 24 (2 PrSMs) Without: 22, with: 25 (1 PrSM) Without: 22, with: 26 (2 PrSMs) Without: 22, with: 27 (6 PrSMs) Without: 22, with: 28 (5 PrSMs) Without: 22, with: 29 (5 PrSMs) Without: 22, with: 30 (7 PrSMs) Without: 22, with: 31 (9 PrSMs) Without: 22, with: 32 (8 PrSMs) Without: 22, with: 33 (5 PrSMs) Without: 22, with: 34 (5 PrSMs) Without: 22, with: 35 (3 PrSMs) Without: 22, with: 36 (6 PrSMs) Without: 22, with: 37 (3 PrSMs) Without: 22, with: 39 (1 PrSM) Without: 22, with: 40 (2 PrSMs) Without: 22, with: 42 (2 PrSMs) Without: 23, with: 25 (2 PrSMs) Without: 23, with: 26 (1 PrSM) Without: 23, with: 27 (2 PrSMs) Without: 23, with: 28 (3 PrSMs) Without: 23, with: 29 (7 PrSMs) Without: 23, with: 30 (4 PrSMs) Without: 23, with: 31 (12 PrSMs) Without: 23, with: 32 (6 PrSMs) Without: 23, with: 33 (7 PrSMs) Without: 23, with: 34 (5 PrSMs) Without: 23, with: 35 (7 PrSMs) Without: 23, with: 36 (6 PrSMs) Without: 23, with: 37 (2 PrSMs) Without: 23, with: 38 (3 PrSMs) Without: 23, with: 42 (1 PrSM) Without: 24, with: 25 (1 PrSM) Without: 24, with: 26 (1 PrSM) Without: 24, with: 27 (4 PrSMs) Without: 24, with: 28 (3 PrSMs) Without: 24, with: 29 (7 PrSMs) Without: 24, with: 30 (5 PrSMs) Without: 24, with: 31 (12 PrSMs) Without: 24, with: 32 (8 PrSMs) Without: 24, with: 33 (7 PrSMs) Without: 24, with: 34 (8 PrSMs) Without: 24, with: 35 (3 PrSMs) Without: 24, with: 36 (7 PrSMs) Without: 24, with: 37 (8 PrSMs) Without: 24, with: 38 (4 PrSMs) Without: 24, with: 39 (1 PrSM) Without: 24, with: 40 (3 PrSMs) Without: 24, with: 41 (5 PrSMs) Without: 24, with: 55 (1 PrSM) Without: 25, with: 28 (4 PrSMs) Without: 25, with: 29 (4 PrSMs) Without: 25, with: 30 (4 PrSMs) Without: 25, with: 31 (2 PrSMs) Without: 25, with: 32 (5 PrSMs) Without: 25, with: 33 (6 PrSMs) Without: 25, with: 34 (6 PrSMs) Without: 25, with: 35 (2 PrSMs) Without: 25, with: 36 (6 PrSMs) Without: 25, with: 37 (12 PrSMs) Without: 25, with: 38 (5 PrSMs) Without: 25, with: 39 (2 PrSMs) Without: 25, with: 40 (6 PrSMs) Without: 25, with: 41 (5 PrSMs) Without: 25, with: 42 (2 PrSMs) Without: 25, with: 43 (1 PrSM) Without: 25, with: 44 (3 PrSMs) Without: 25, with: 45 (1 PrSM) Without: 25, with: 47 (1 PrSM) Without: 26, with: 29 (3 PrSMs) Without: 26, with: 30 (3 PrSMs) Without: 26, with: 31 (8 PrSMs) Without: 26, with: 32 (8 PrSMs) Without: 26, with: 33 (6 PrSMs) Without: 26, with: 34 (7 PrSMs) Without: 26, with: 35 (8 PrSMs) Without: 26, with: 36 (5 PrSMs) Without: 26, with: 37 (7 PrSMs) Without: 26, with: 38 (10 PrSMs) Without: 26, with: 39 (4 PrSMs) Without: 26, with: 40 (1 PrSM) Without: 26, with: 42 (1 PrSM) Without: 26, with: 43 (1 PrSM) Without: 27, with: 30 (1 PrSM) Without: 27, with: 31 (3 PrSMs) Without: 27, with: 32 (8 PrSMs) Without: 27, with: 33 (2 PrSMs) Without: 27, with: 34 (7 PrSMs) Without: 27, with: 35 (7 PrSMs) Without: 27, with: 36 (5 PrSMs) Without: 27, with: 37 (4 PrSMs) Without: 27, with: 38 (7 PrSMs) Without: 27, with: 39 (7 PrSMs) Without: 27, with: 40 (9 PrSMs) Without: 27, with: 41 (2 PrSMs) Without: 27, with: 42 (4 PrSMs) Without: 27, with: 43 (2 PrSMs) Without: 27, with: 44 (1 PrSM) Without: 27, with: 45 (3 PrSMs) Without: 27, with: 46 (1 PrSM) Without: 27, with: 47 (1 PrSM) Without: 27, with: 50 (1 PrSM) Without: 28, with: 29 (1 PrSM) Without: 28, with: 30 (1 PrSM) Without: 28, with: 32 (6 PrSMs) Without: 28, with: 33 (5 PrSMs) Without: 28, with: 34 (7 PrSMs) Without: 28, with: 35 (5 PrSMs) Without: 28, with: 36 (10 PrSMs) Without: 28, with: 37 (7 PrSMs) Without: 28, with: 38 (10 PrSMs) Without: 28, with: 39 (1 PrSM) Without: 28, with: 40 (6 PrSMs) Without: 28, with: 41 (1 PrSM) Without: 28, with: 42 (5 PrSMs) Without: 28, with: 43 (2 PrSMs) Without: 28, with: 44 (1 PrSM) Without: 28, with: 46 (1 PrSM) Without: 28, with: 48 (1 PrSM) Without: 28, with: 49 (2 PrSMs) Without: 28, with: 52 (1 PrSM) Without: 29, with: 30 (1 PrSM) Without: 29, with: 31 (1 PrSM) Without: 29, with: 32 (1 PrSM) Without: 29, with: 34 (3 PrSMs) Without: 29, with: 35 (1 PrSM) Without: 29, with: 36 (7 PrSMs) Without: 29, with: 37 (5 PrSMs) Without: 29, with: 38 (8 PrSMs) Without: 29, with: 39 (7 PrSMs) Without: 29, with: 40 (5 PrSMs) Without: 29, with: 41 (4 PrSMs) Without: 29, with: 42 (7 PrSMs) Without: 29, with: 43 (6 PrSMs) Without: 29, with: 44 (2 PrSMs) Without: 29, with: 45 (3 PrSMs) Without: 29, with: 46 (2 PrSMs) Without: 29, with: 47 (1 PrSM) Without: 29, with: 50 (1 PrSM) Without: 30, with: 32 (1 PrSM) Without: 30, with: 35 (4 PrSMs) Without: 30, with: 36 (8 PrSMs) Without: 30, with: 37 (5 PrSMs) Without: 30, with: 38 (2 PrSMs) Without: 30, with: 39 (9 PrSMs) Without: 30, with: 40 (2 PrSMs) Without: 30, with: 41 (7 PrSMs) Without: 30, with: 42 (2 PrSMs) Without: 30, with: 43 (3 PrSMs) Without: 30, with: 45 (5 PrSMs) Without: 30, with: 46 (2 PrSMs) Without: 30, with: 47 (4 PrSMs) Without: 30, with: 49 (1 PrSM) Without: 30, with: 50 (1 PrSM) Without: 30, with: 52 (1 PrSM) Without: 30, with: 63 (1 PrSM) Without: 31, with: 33 (1 PrSM) Without: 31, with: 36 (3 PrSMs) Without: 31, with: 37 (1 PrSM) Without: 31, with: 38 (3 PrSMs) Without: 31, with: 39 (4 PrSMs) Without: 31, with: 40 (6 PrSMs) Without: 31, with: 41 (4 PrSMs) Without: 31, with: 42 (6 PrSMs) Without: 31, with: 43 (2 PrSMs) Without: 31, with: 44 (7 PrSMs) Without: 31, with: 45 (5 PrSMs) Without: 31, with: 46 (4 PrSMs) Without: 31, with: 48 (3 PrSMs) Without: 31, with: 49 (1 PrSM) Without: 31, with: 50 (1 PrSM) Without: 31, with: 52 (1 PrSM) Without: 32, with: 36 (2 PrSMs) Without: 32, with: 37 (1 PrSM) Without: 32, with: 38 (6 PrSMs) Without: 32, with: 39 (6 PrSMs) Without: 32, with: 40 (7 PrSMs) Without: 32, with: 41 (8 PrSMs) Without: 32, with: 42 (8 PrSMs) Without: 32, with: 43 (2 PrSMs) Without: 32, with: 44 (6 PrSMs) Without: 32, with: 45 (8 PrSMs) Without: 32, with: 46 (3 PrSMs) Without: 32, with: 47 (3 PrSMs) Without: 32, with: 48 (2 PrSMs) Without: 32, with: 49 (1 PrSM) Without: 32, with: 50 (5 PrSMs) Without: 32, with: 51 (1 PrSM) Without: 33, with: 35 (1 PrSM) Without: 33, with: 36 (1 PrSM) Without: 33, with: 37 (1 PrSM) Without: 33, with: 38 (4 PrSMs) Without: 33, with: 39 (7 PrSMs) Without: 33, with: 40 (9 PrSMs) Without: 33, with: 41 (1 PrSM) Without: 33, with: 42 (6 PrSMs) Without: 33, with: 43 (12 PrSMs) Without: 33, with: 44 (4 PrSMs) Without: 33, with: 45 (4 PrSMs) Without: 33, with: 46 (7 PrSMs) Without: 33, with: 47 (5 PrSMs) Without: 33, with: 48 (1 PrSM) Without: 33, with: 49 (5 PrSMs) Without: 33, with: 50 (2 PrSMs) Without: 33, with: 51 (2 PrSMs) Without: 33, with: 52 (5 PrSMs) Without: 33, with: 53 (1 PrSM) Without: 33, with: 58 (1 PrSM) Without: 33, with: 59 (1 PrSM) Without: 33, with: 60 (2 PrSMs) Without: 34, with: 36 (1 PrSM) Without: 34, with: 37 (3 PrSMs) Without: 34, with: 38 (2 PrSMs) Without: 34, with: 39 (2 PrSMs) Without: 34, with: 40 (3 PrSMs) Without: 34, with: 41 (2 PrSMs) Without: 34, with: 42 (12 PrSMs) Without: 34, with: 43 (4 PrSMs) Without: 34, with: 44 (5 PrSMs) Without: 34, with: 45 (6 PrSMs) Without: 34, with: 46 (6 PrSMs) Without: 34, with: 47 (2 PrSMs) Without: 34, with: 48 (5 PrSMs) Without: 34, with: 49 (4 PrSMs) Without: 34, with: 50 (2 PrSMs) Without: 34, with: 53 (1 PrSM) Without: 34, with: 54 (1 PrSM) Without: 35, with: 36 (1 PrSM) Without: 35, with: 37 (1 PrSM) Without: 35, with: 38 (2 PrSMs) Without: 35, with: 39 (5 PrSMs) Without: 35, with: 40 (7 PrSMs) Without: 35, with: 41 (4 PrSMs) Without: 35, with: 42 (4 PrSMs) Without: 35, with: 43 (5 PrSMs) Without: 35, with: 44 (10 PrSMs) Without: 35, with: 45 (2 PrSMs) Without: 35, with: 46 (3 PrSMs) Without: 35, with: 47 (4 PrSMs) Without: 35, with: 48 (5 PrSMs) Without: 35, with: 49 (4 PrSMs) Without: 35, with: 50 (3 PrSMs) Without: 35, with: 52 (2 PrSMs) Without: 35, with: 53 (2 PrSMs) Without: 35, with: 55 (1 PrSM) Without: 35, with: 58 (1 PrSM) Without: 36, with: 41 (2 PrSMs) Without: 36, with: 42 (6 PrSMs) Without: 36, with: 43 (4 PrSMs) Without: 36, with: 44 (6 PrSMs) Without: 36, with: 45 (5 PrSMs) Without: 36, with: 46 (3 PrSMs) Without: 36, with: 47 (4 PrSMs) Without: 36, with: 48 (4 PrSMs) Without: 36, with: 49 (3 PrSMs) Without: 36, with: 50 (1 PrSM) Without: 36, with: 51 (4 PrSMs) Without: 36, with: 52 (3 PrSMs) Without: 36, with: 54 (1 PrSM) Without: 36, with: 57 (2 PrSMs) Without: 36, with: 58 (1 PrSM) Without: 37, with: 42 (2 PrSMs) Without: 37, with: 43 (4 PrSMs) Without: 37, with: 44 (7 PrSMs) Without: 37, with: 45 (4 PrSMs) Without: 37, with: 46 (4 PrSMs) Without: 37, with: 47 (9 PrSMs) Without: 37, with: 48 (2 PrSMs) Without: 37, with: 49 (5 PrSMs) Without: 37, with: 50 (4 PrSMs) Without: 37, with: 51 (2 PrSMs) Without: 37, with: 52 (1 PrSM) Without: 37, with: 53 (3 PrSMs) Without: 37, with: 54 (3 PrSMs) Without: 37, with: 56 (2 PrSMs) Without: 37, with: 58 (1 PrSM) Without: 37, with: 59 (1 PrSM) Without: 37, with: 62 (1 PrSM) Without: 38, with: 41 (1 PrSM) Without: 38, with: 44 (4 PrSMs) Without: 38, with: 45 (4 PrSMs) Without: 38, with: 46 (6 PrSMs) Without: 38, with: 47 (9 PrSMs) Without: 38, with: 48 (1 PrSM) Without: 38, with: 49 (2 PrSMs) Without: 38, with: 50 (5 PrSMs) Without: 38, with: 51 (3 PrSMs) Without: 38, with: 52 (1 PrSM) Without: 38, with: 53 (3 PrSMs) Without: 38, with: 55 (2 PrSMs) Without: 38, with: 56 (1 PrSM) Without: 38, with: 57 (1 PrSM) Without: 38, with: 59 (1 PrSM) Without: 39, with: 42 (1 PrSM) Without: 39, with: 44 (1 PrSM) Without: 39, with: 45 (6 PrSMs) Without: 39, with: 46 (5 PrSMs) Without: 39, with: 47 (6 PrSMs) Without: 39, with: 48 (1 PrSM) Without: 39, with: 49 (3 PrSMs) Without: 39, with: 50 (4 PrSMs) Without: 39, with: 51 (2 PrSMs) Without: 39, with: 52 (4 PrSMs) Without: 39, with: 53 (4 PrSMs) Without: 39, with: 54 (3 PrSMs) Without: 39, with: 55 (3 PrSMs) Without: 39, with: 56 (1 PrSM) Without: 39, with: 57 (3 PrSMs) Without: 39, with: 58 (1 PrSM) Without: 39, with: 59 (1 PrSM) Without: 39, with: 61 (2 PrSMs) Without: 39, with: 64 (1 PrSM) Without: 40, with: 43 (1 PrSM) Without: 40, with: 45 (4 PrSMs) Without: 40, with: 47 (3 PrSMs) Without: 40, with: 48 (3 PrSMs) Without: 40, with: 49 (5 PrSMs) Without: 40, with: 50 (2 PrSMs) Without: 40, with: 51 (2 PrSMs) Without: 40, with: 52 (1 PrSM) Without: 40, with: 53 (4 PrSMs) Without: 40, with: 54 (3 PrSMs) Without: 40, with: 55 (1 PrSM) Without: 40, with: 57 (1 PrSM) Without: 40, with: 59 (1 PrSM) Without: 40, with: 60 (1 PrSM) Without: 41, with: 44 (2 PrSMs) Without: 41, with: 45 (1 PrSM) Without: 41, with: 46 (2 PrSMs) Without: 41, with: 47 (1 PrSM) Without: 41, with: 48 (3 PrSMs) Without: 41, with: 49 (1 PrSM) Without: 41, with: 50 (4 PrSMs) Without: 41, with: 51 (6 PrSMs) Without: 41, with: 52 (5 PrSMs) Without: 41, with: 53 (4 PrSMs) Without: 41, with: 54 (3 PrSMs) Without: 41, with: 55 (3 PrSMs) Without: 41, with: 56 (3 PrSMs) Without: 41, with: 58 (1 PrSM) Without: 41, with: 61 (1 PrSM) Without: 41, with: 62 (1 PrSM) Without: 41, with: 63 (1 PrSM) Without: 41, with: 64 (1 PrSM) Without: 41, with: 71 (1 PrSM) Without: 42, with: 47 (1 PrSM) Without: 42, with: 48 (2 PrSMs) Without: 42, with: 50 (1 PrSM) Without: 42, with: 52 (2 PrSMs) Without: 42, with: 53 (3 PrSMs) Without: 42, with: 54 (4 PrSMs) Without: 42, with: 55 (6 PrSMs) Without: 42, with: 56 (1 PrSM) Without: 42, with: 57 (1 PrSM) Without: 42, with: 59 (1 PrSM) Without: 42, with: 63 (1 PrSM) Without: 42, with: 70 (1 PrSM) Without: 43, with: 49 (1 PrSM) Without: 43, with: 50 (2 PrSMs) Without: 43, with: 51 (5 PrSMs) Without: 43, with: 52 (7 PrSMs) Without: 43, with: 53 (2 PrSMs) Without: 43, with: 54 (3 PrSMs) Without: 43, with: 55 (2 PrSMs) Without: 43, with: 56 (3 PrSMs) Without: 43, with: 57 (1 PrSM) Without: 43, with: 58 (1 PrSM) Without: 43, with: 59 (1 PrSM) Without: 43, with: 61 (2 PrSMs) Without: 43, with: 62 (1 PrSM) Without: 43, with: 65 (1 PrSM) Without: 44, with: 45 (2 PrSMs) Without: 44, with: 47 (1 PrSM) Without: 44, with: 48 (2 PrSMs) Without: 44, with: 49 (1 PrSM) Without: 44, with: 50 (2 PrSMs) Without: 44, with: 51 (4 PrSMs) Without: 44, with: 52 (3 PrSMs) Without: 44, with: 53 (2 PrSMs) Without: 44, with: 54 (1 PrSM) Without: 44, with: 55 (2 PrSMs) Without: 44, with: 56 (3 PrSMs) Without: 44, with: 57 (2 PrSMs) Without: 44, with: 58 (1 PrSM) Without: 44, with: 60 (3 PrSMs) Without: 44, with: 61 (2 PrSMs) Without: 44, with: 63 (1 PrSM) Without: 44, with: 64 (2 PrSMs) Without: 44, with: 69 (1 PrSM) Without: 45, with: 47 (1 PrSM) Without: 45, with: 48 (1 PrSM) Without: 45, with: 49 (1 PrSM) Without: 45, with: 50 (2 PrSMs) Without: 45, with: 51 (5 PrSMs) Without: 45, with: 52 (1 PrSM) Without: 45, with: 53 (2 PrSMs) Without: 45, with: 54 (1 PrSM) Without: 45, with: 55 (2 PrSMs) Without: 45, with: 56 (5 PrSMs) Without: 45, with: 57 (1 PrSM) Without: 45, with: 58 (1 PrSM) Without: 45, with: 59 (1 PrSM) Without: 45, with: 60 (3 PrSMs) Without: 45, with: 61 (1 PrSM) Without: 45, with: 62 (2 PrSMs) Without: 45, with: 63 (1 PrSM) Without: 45, with: 64 (2 PrSMs) Without: 45, with: 66 (3 PrSMs) Without: 46, with: 50 (1 PrSM) Without: 46, with: 52 (3 PrSMs) Without: 46, with: 53 (4 PrSMs) Without: 46, with: 54 (1 PrSM) Without: 46, with: 55 (2 PrSMs) Without: 46, with: 56 (3 PrSMs) Without: 46, with: 58 (4 PrSMs) Without: 46, with: 59 (1 PrSM) Without: 46, with: 60 (2 PrSMs) Without: 46, with: 61 (2 PrSMs) Without: 46, with: 64 (1 PrSM) Without: 46, with: 67 (1 PrSM) Without: 47, with: 50 (1 PrSM) Without: 47, with: 53 (6 PrSMs) Without: 47, with: 55 (1 PrSM) Without: 47, with: 57 (4 PrSMs) Without: 47, with: 59 (2 PrSMs) Without: 47, with: 60 (1 PrSM) Without: 47, with: 62 (1 PrSM) Without: 47, with: 63 (1 PrSM) Without: 47, with: 65 (1 PrSM) Without: 47, with: 67 (1 PrSM) Without: 48, with: 51 (1 PrSM) Without: 48, with: 54 (3 PrSMs) Without: 48, with: 55 (1 PrSM) Without: 48, with: 56 (2 PrSMs) Without: 48, with: 57 (6 PrSMs) Without: 48, with: 59 (1 PrSM) Without: 48, with: 60 (3 PrSMs) Without: 48, with: 61 (1 PrSM) Without: 48, with: 62 (1 PrSM) Without: 48, with: 63 (2 PrSMs) Without: 48, with: 65 (1 PrSM) Without: 48, with: 66 (1 PrSM) Without: 48, with: 67 (1 PrSM) Without: 48, with: 71 (1 PrSM) Without: 49, with: 54 (5 PrSMs) Without: 49, with: 56 (1 PrSM) Without: 49, with: 57 (1 PrSM) Without: 49, with: 58 (6 PrSMs) Without: 49, with: 59 (1 PrSM) Without: 49, with: 60 (3 PrSMs) Without: 49, with: 62 (3 PrSMs) Without: 49, with: 63 (1 PrSM) Without: 49, with: 64 (1 PrSM) Without: 49, with: 65 (1 PrSM) Without: 49, with: 82 (1 PrSM) Without: 50, with: 54 (1 PrSM) Without: 50, with: 55 (1 PrSM) Without: 50, with: 56 (1 PrSM) Without: 50, with: 57 (1 PrSM) Without: 50, with: 59 (2 PrSMs) Without: 50, with: 61 (1 PrSM) Without: 50, with: 62 (2 PrSMs) Without: 50, with: 64 (1 PrSM) Without: 50, with: 66 (1 PrSM) Without: 50, with: 67 (2 PrSMs) Without: 51, with: 53 (1 PrSM) Without: 51, with: 57 (2 PrSMs) Without: 51, with: 58 (3 PrSMs) Without: 51, with: 59 (3 PrSMs) Without: 51, with: 60 (2 PrSMs) Without: 51, with: 61 (4 PrSMs) Without: 51, with: 64 (2 PrSMs) Without: 51, with: 68 (2 PrSMs) Without: 51, with: 69 (1 PrSM) Without: 51, with: 74 (1 PrSM) Without: 52, with: 55 (1 PrSM) Without: 52, with: 56 (1 PrSM) Without: 52, with: 57 (2 PrSMs) Without: 52, with: 58 (1 PrSM) Without: 52, with: 61 (2 PrSMs) Without: 52, with: 62 (1 PrSM) Without: 52, with: 63 (1 PrSM) Without: 52, with: 65 (3 PrSMs) Without: 52, with: 66 (2 PrSMs) Without: 52, with: 68 (2 PrSMs) Without: 53, with: 58 (1 PrSM) Without: 53, with: 59 (2 PrSMs) Without: 53, with: 60 (2 PrSMs) Without: 53, with: 61 (1 PrSM) Without: 53, with: 62 (1 PrSM) Without: 53, with: 63 (1 PrSM) Without: 53, with: 64 (3 PrSMs) Without: 53, with: 65 (3 PrSMs) Without: 53, with: 67 (1 PrSM) Without: 53, with: 69 (1 PrSM) Without: 53, with: 73 (1 PrSM) Without: 53, with: 74 (1 PrSM) Without: 54, with: 58 (2 PrSMs) Without: 54, with: 60 (1 PrSM) Without: 54, with: 61 (2 PrSMs) Without: 54, with: 64 (3 PrSMs) Without: 54, with: 65 (4 PrSMs) Without: 54, with: 66 (3 PrSMs) Without: 54, with: 67 (2 PrSMs) Without: 54, with: 70 (2 PrSMs) Without: 54, with: 76 (1 PrSM) Without: 55, with: 58 (1 PrSM) Without: 55, with: 59 (1 PrSM) Without: 55, with: 60 (1 PrSM) Without: 55, with: 63 (2 PrSMs) Without: 55, with: 65 (1 PrSM) Without: 55, with: 66 (1 PrSM) Without: 55, with: 68 (1 PrSM) Without: 55, with: 69 (1 PrSM) Without: 55, with: 79 (1 PrSM) Without: 56, with: 62 (1 PrSM) Without: 56, with: 63 (1 PrSM) Without: 56, with: 64 (1 PrSM) Without: 56, with: 65 (1 PrSM) Without: 56, with: 66 (2 PrSMs) Without: 56, with: 67 (2 PrSMs) Without: 56, with: 68 (1 PrSM) Without: 56, with: 69 (1 PrSM) Without: 56, with: 72 (2 PrSMs) Without: 56, with: 92 (1 PrSM) Without: 57, with: 63 (1 PrSM) Without: 57, with: 64 (1 PrSM) Without: 57, with: 65 (1 PrSM) Without: 57, with: 66 (3 PrSMs) Without: 57, with: 67 (1 PrSM) Without: 57, with: 68 (1 PrSM) Without: 57, with: 69 (1 PrSM) Without: 57, with: 71 (1 PrSM) Without: 57, with: 72 (2 PrSMs) Without: 57, with: 73 (1 PrSM) Without: 57, with: 76 (1 PrSM) Without: 58, with: 62 (1 PrSM) Without: 58, with: 64 (1 PrSM) Without: 58, with: 65 (1 PrSM) Without: 58, with: 66 (1 PrSM) Without: 58, with: 67 (2 PrSMs) Without: 58, with: 68 (1 PrSM) Without: 58, with: 69 (1 PrSM) Without: 58, with: 73 (1 PrSM) Without: 58, with: 74 (1 PrSM) Without: 59, with: 62 (1 PrSM) Without: 59, with: 66 (2 PrSMs) Without: 59, with: 67 (1 PrSM) Without: 59, with: 69 (2 PrSMs) Without: 59, with: 71 (2 PrSMs) Without: 59, with: 72 (1 PrSM) Without: 59, with: 73 (2 PrSMs) Without: 59, with: 76 (1 PrSM) Without: 59, with: 78 (1 PrSM) Without: 59, with: 80 (1 PrSM) Without: 60, with: 69 (1 PrSM) Without: 60, with: 70 (1 PrSM) Without: 60, with: 71 (2 PrSMs) Without: 60, with: 74 (1 PrSM) Without: 60, with: 75 (2 PrSMs) Without: 60, with: 84 (1 PrSM) Without: 61, with: 67 (1 PrSM) Without: 61, with: 68 (1 PrSM) Without: 61, with: 69 (2 PrSMs) Without: 61, with: 71 (1 PrSM) Without: 61, with: 78 (2 PrSMs) Without: 62, with: 66 (1 PrSM) Without: 62, with: 70 (2 PrSMs) Without: 62, with: 71 (2 PrSMs) Without: 62, with: 73 (1 PrSM) Without: 62, with: 75 (1 PrSM) Without: 62, with: 77 (2 PrSMs) Without: 62, with: 83 (1 PrSM) Without: 63, with: 70 (1 PrSM) Without: 63, with: 71 (1 PrSM) Without: 63, with: 72 (1 PrSM) Without: 63, with: 77 (1 PrSM) Without: 63, with: 78 (1 PrSM) Without: 63, with: 81 (1 PrSM) Without: 63, with: 83 (1 PrSM) Without: 63, with: 86 (1 PrSM) Without: 64, with: 69 (1 PrSM) Without: 64, with: 70 (1 PrSM) Without: 64, with: 73 (1 PrSM) Without: 64, with: 76 (1 PrSM) Without: 64, with: 83 (1 PrSM) Without: 65, with: 75 (1 PrSM) Without: 65, with: 76 (2 PrSMs) Without: 65, with: 81 (1 PrSM) Without: 65, with: 82 (1 PrSM) Without: 66, with: 74 (2 PrSMs) Without: 66, with: 75 (1 PrSM) Without: 66, with: 87 (2 PrSMs) Without: 67, with: 70 (1 PrSM) Without: 67, with: 72 (1 PrSM) Without: 67, with: 75 (1 PrSM) Without: 67, with: 81 (1 PrSM) Without: 67, with: 87 (1 PrSM) Without: 69, with: 76 (1 PrSM) Without: 69, with: 79 (1 PrSM) Without: 69, with: 80 (1 PrSM) Without: 69, with: 89 (1 PrSM) Without: 70, with: 77 (1 PrSM) Without: 70, with: 78 (1 PrSM) Without: 70, with: 80 (1 PrSM) Without: 71, with: 79 (1 PrSM) Without: 71, with: 84 (1 PrSM) Without: 72, with: 82 (1 PrSM) Without: 73, with: 86 (1 PrSM) Without: 73, with: 89 (1 PrSM) Without: 74, with: 89 (1 PrSM) Without: 75, with: 82 (1 PrSM) Without: 75, with: 83 (1 PrSM) Without: 75, with: 84 (1 PrSM) Without: 76, with: 84 (1 PrSM) Without: 76, with: 89 (1 PrSM) Without: 77, with: 85 (1 PrSM) Without: 77, with: 92 (1 PrSM) Without: 77, with: 99 (1 PrSM) Without: 78, with: 88 (1 PrSM) Without: 79, with: 90 (1 PrSM) Without: 79, with: 92 (1 PrSM) Without: 81, with: 98 (1 PrSM) Without: 83, with: 109 (1 PrSM) Without: 86, with: 100 (1 PrSM) Without: 88, with: 94 (1 PrSM) Without: 88, with: 102 (1 PrSM) Without: 88, with: 106 (1 PrSM) Without: 94, with: 108 (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 spectrum170180
Matched fragment masses2434
Matched fragment ions2030

Post mass matching added a median of 9 matched fragment masses per PrSM (interquartile range 6 to 12, maximum 36). The number of matched fragment ions per PrSM rose from a median of 20 to 30. The added masses are mostly small (median 1.21 kDa) and carry low charges (76% are singly or doubly charged).

The two runs report the same 2,966 PrSMs, 1,765 proteoforms, and 416 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. It adds 0.5 minutes to the 5.8-minute search.