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).
  • 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.
  • Computer: a Linux workstation with 20 CPU cores and 62 GB of RAM.

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

This benchmark compares the identification results of TopFD and TopPIC in TopPIC Suite versions 1.8.1 and 1.9.0 with their default settings: at most one unexpected mass shift and no variable modifications.

Settings

Version 1.8.1Version 1.9.0
TopFD commandtopfd -g 480_F3_02.mzMLtopfd -N 480_F3_02.mzML
TopPIC commandtoppic -f C57 -g -d -t FDR -T FDR db.fasta 480_F3_02_ms2.msaligntoppic -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; version 1.9.0 no longer generates them. The option -N in version 1.9.0 skips the SQLite output. Neither option changes the identification results.

Results

Identifications at 1% FDR Identifications at 1% FDR Version 1.8.1 Version 1.9.0 0 1,000 2,000 3,000 Number of identifications PrSMs, version 1.8.1: 2,797 2,797 PrSMs, version 1.9.0: 2,873 2,873 PrSMs Proteoforms, version 1.8.1: 1,655 1,655 Proteoforms, version 1.9.0: 1,686 1,686 Proteoforms
Version 1.8.1Version 1.9.0Change
PrSMs (1% spectrum-level FDR)2,7972,873+2.7%
Proteoforms (1% proteoform-level FDR)1,6551,686+1.9%

Version 1.9.0 identified 2.7% more PrSMs and 1.9% more proteoforms than version 1.8.1.

3 TopPIC 1.9.0 search settings

This benchmark compares four search settings of TopPIC 1.9.0 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 up to two unexpected mass shifts (-s 2), a search with methionine oxidation as a variable modification (-b var_mods.txt), 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-s 2At most 2 unexpected mass shifts
c-b var_mods.txtAt most 1 unexpected mass shift, variable oxidation on M (at most 3 sites)
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 20 40 60 80 100 120 140 Wall-clock time (minutes) a: default, TopPIC: 48.6 minutes 48.6 a: default b: -s 2, TopPIC: 124.1 minutes 124.1 b: -s 2 c: -b, TopPIC: 54.4 minutes 54.4 c: -b d: -s 2 -b, TopPIC: 121.4 minutes 121.4 d: -s 2 -b
Identifications at 1% FDR Identifications at 1% FDR PrSMs Proteoforms 0 500 1,000 1,500 2,000 2,500 3,000 3,500 Number of identifications a: default, PrSMs: 2,873 2,873 a: default, Proteoforms: 1,686 1,686 a: default b: -s 2, PrSMs: 2,974 2,974 b: -s 2, Proteoforms: 1,744 1,744 b: -s 2 c: -b, PrSMs: 2,927 2,927 c: -b, Proteoforms: 1,727 1,727 c: -b d: -s 2 -b, PrSMs: 3,027 3,027 d: -s 2 -b, Proteoforms: 1,779 1,779 d: -s 2 -b
SettingRunning time (minutes)Peak memoryPrSMsProteoformsProteins
a: default48.611.6 GB2,8731,686393
b: -s 2124.111.7 GB2,974 (+3.5%)1,744 (+3.4%)395
c: -b var_mods.txt54.416.0 GB2,927 (+1.9%)1,727 (+2.4%)396
d: -s 2 -b var_mods.txt121.416.0 GB3,027 (+5.4%)1,779 (+5.5%)398

Running time

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 from 48.6 to about 2 hours (2.5 times longer). Variable oxidation (-b) adds only about 6 minutes and 4 GB of memory.

Identifications

Every setting is a superset of the default search: no PrSM identified by the default setting is lost in settings b, c, or d, and no shared E-value gets worse. Setting b adds 101 PrSMs and 58 proteoforms, mainly proteoforms with two unknown alterations that the one-shift search cannot explain. Setting c adds 54 PrSMs and 41 proteoforms, and it also re-explains 283 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. The two options are nearly independent, and setting d gives the largest set: 3,027 PrSMs and 1,779 proteoforms, 5.4% and 5.5% more than the default setting. Protein counts change by at most 5, 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 little and gives more accurate proteoform assignments. Add -s 2 when running time allows.

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. TopPIC 1.9 added post mass matching, which matches theoretical fragment ions directly to centroided peaks after proteoform identification 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 (-E). After the database search and E-value computation, each theoretical fragment mass of a PrSM 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 step does not change the identifications, E-values, or FDR; it only adds matched fragment masses.

Settings

Without post mass matchingWith post mass matching
TopFD commandtopfd -N 480_F3_02.mzMLtopfd 480_F3_02.mzML
TopPIC commandtoppic -f C57 -d -t FDR -T FDR -y FDR -E db.fasta 480_F3_02_ms2.msaligntoppic -f C57 -d -t FDR -T FDR -y FDR db.fasta 480_F3_02_ms2.msalign

The run without post mass matching is setting a of benchmark 3, which was run before post mass matching was added to TopPIC and is equivalent to a run with -E. Both runs use the default search settings of TopPIC 1.9.0 and identify almost the same PrSMs. Post mass matching reads the centroided peaks from the SQLite file that TopFD writes when it is run without -N.

Results

Distribution of matched fragment masses per PrSM Matched fragment masses per PrSM Without post mass matching With post mass matching 0 50 100 150 200 250 300 350 400 450 500 550 Number of PrSMs 0–4 matched masses, without post mass matching: 3 PrSMs 0–4 matched masses, with post mass matching: 2 PrSMs 0–4 5–9 matched masses, without post mass matching: 241 PrSMs 5–9 matched masses, with post mass matching: 26 PrSMs 5–9 10–14 matched masses, without post mass matching: 455 PrSMs 10–14 matched masses, with post mass matching: 104 PrSMs 10–14 15–19 matched masses, without post mass matching: 522 PrSMs 15–19 matched masses, with post mass matching: 267 PrSMs 15–19 20–24 matched masses, without post mass matching: 436 PrSMs 20–24 matched masses, with post mass matching: 325 PrSMs 20–24 25–29 matched masses, without post mass matching: 366 PrSMs 25–29 matched masses, with post mass matching: 332 PrSMs 25–29 30–34 matched masses, without post mass matching: 300 PrSMs 30–34 matched masses, with post mass matching: 401 PrSMs 30–34 35–39 matched masses, without post mass matching: 171 PrSMs 35–39 matched masses, with post mass matching: 345 PrSMs 35–39 40–44 matched masses, without post mass matching: 144 PrSMs 40–44 matched masses, with post mass matching: 306 PrSMs 40–44 45–49 matched masses, without post mass matching: 97 PrSMs 45–49 matched masses, with post mass matching: 224 PrSMs 45–49 50–54 matched masses, without post mass matching: 52 PrSMs 50–54 matched masses, with post mass matching: 171 PrSMs 50–54 55–59 matched masses, without post mass matching: 27 PrSMs 55–59 matched masses, with post mass matching: 120 PrSMs 55–59 60–64 matched masses, without post mass matching: 17 PrSMs 60–64 matched masses, with post mass matching: 93 PrSMs 60–64 65–69 matched masses, without post mass matching: 12 PrSMs 65–69 matched masses, with post mass matching: 55 PrSMs 65–69 70–74 matched masses, without post mass matching: 4 PrSMs 70–74 matched masses, with post mass matching: 29 PrSMs 70–74 75–79 matched masses, without post mass matching: 3 PrSMs 75–79 matched masses, with post mass matching: 23 PrSMs 75–79 80–84 matched masses, without post mass matching: 3 PrSMs 80–84 matched masses, with post mass matching: 11 PrSMs 80–84 85–89 matched masses, with post mass matching: 10 PrSMs 85–89 90–94 matched masses, with post mass matching: 4 PrSMs 90–94 95–99 matched masses, with post mass matching: 1 PrSMs 95–99 100–104 matched masses, with post mass matching: 4 PrSMs 100–104 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: 5 (1 PrSM) Without: 5, with: 5 (4 PrSMs) Without: 5, with: 6 (3 PrSMs) Without: 5, with: 8 (1 PrSM) Without: 5, with: 9 (1 PrSM) Without: 5, with: 10 (3 PrSMs) Without: 5, with: 11 (1 PrSM) Without: 5, with: 14 (1 PrSM) Without: 5, with: 17 (1 PrSM) Without: 6, with: 6 (2 PrSMs) Without: 6, with: 8 (1 PrSM) Without: 6, with: 9 (2 PrSMs) Without: 6, with: 10 (2 PrSMs) Without: 6, with: 11 (3 PrSMs) Without: 6, with: 12 (3 PrSMs) Without: 6, with: 13 (4 PrSMs) Without: 6, with: 14 (2 PrSMs) Without: 6, with: 15 (4 PrSMs) Without: 6, with: 16 (5 PrSMs) Without: 6, with: 17 (2 PrSMs) Without: 6, with: 18 (2 PrSMs) Without: 6, with: 19 (3 PrSMs) Without: 6, with: 20 (2 PrSMs) Without: 6, with: 21 (1 PrSM) Without: 6, with: 22 (1 PrSM) Without: 6, with: 24 (1 PrSM) Without: 6, with: 29 (1 PrSM) Without: 7, with: 8 (7 PrSMs) Without: 7, with: 9 (1 PrSM) Without: 7, with: 10 (5 PrSMs) Without: 7, with: 11 (4 PrSMs) Without: 7, with: 12 (5 PrSMs) Without: 7, with: 13 (3 PrSMs) Without: 7, with: 14 (2 PrSMs) Without: 7, with: 15 (6 PrSMs) Without: 7, with: 16 (6 PrSMs) Without: 7, with: 17 (3 PrSMs) Without: 7, with: 18 (2 PrSMs) Without: 7, with: 19 (3 PrSMs) Without: 7, with: 20 (4 PrSMs) Without: 7, with: 21 (4 PrSMs) Without: 7, with: 22 (1 PrSM) Without: 7, with: 23 (2 PrSMs) Without: 7, with: 24 (2 PrSMs) Without: 7, with: 29 (1 PrSM) Without: 8, with: 9 (2 PrSMs) Without: 8, with: 10 (1 PrSM) Without: 8, with: 11 (6 PrSMs) Without: 8, with: 12 (4 PrSMs) Without: 8, with: 13 (4 PrSMs) Without: 8, with: 14 (2 PrSMs) Without: 8, with: 15 (6 PrSMs) Without: 8, with: 16 (4 PrSMs) Without: 8, with: 17 (3 PrSMs) Without: 8, with: 18 (7 PrSMs) Without: 8, with: 19 (6 PrSMs) Without: 8, with: 20 (2 PrSMs) Without: 8, with: 21 (4 PrSMs) Without: 8, with: 24 (1 PrSM) Without: 8, with: 25 (1 PrSM) Without: 9, with: 9 (1 PrSM) Without: 9, with: 10 (5 PrSMs) Without: 9, with: 11 (2 PrSMs) Without: 9, with: 12 (5 PrSMs) Without: 9, with: 13 (7 PrSMs) Without: 9, with: 14 (7 PrSMs) Without: 9, with: 15 (7 PrSMs) Without: 9, with: 16 (5 PrSMs) Without: 9, with: 17 (5 PrSMs) Without: 9, with: 18 (6 PrSMs) Without: 9, with: 19 (4 PrSMs) Without: 9, with: 20 (3 PrSMs) Without: 9, with: 21 (3 PrSMs) Without: 9, with: 22 (1 PrSM) Without: 9, with: 23 (2 PrSMs) Without: 9, with: 24 (2 PrSMs) Without: 9, with: 25 (2 PrSMs) Without: 9, with: 29 (1 PrSM) Without: 9, with: 31 (1 PrSM) Without: 9, with: 32 (1 PrSM) Without: 9, with: 36 (1 PrSM) Without: 10, with: 11 (1 PrSM) Without: 10, with: 12 (3 PrSMs) Without: 10, with: 13 (3 PrSMs) Without: 10, with: 14 (3 PrSMs) Without: 10, with: 15 (9 PrSMs) Without: 10, with: 16 (3 PrSMs) Without: 10, with: 17 (7 PrSMs) Without: 10, with: 18 (6 PrSMs) Without: 10, with: 19 (1 PrSM) Without: 10, with: 20 (2 PrSMs) Without: 10, with: 21 (6 PrSMs) Without: 10, with: 22 (5 PrSMs) Without: 10, with: 23 (7 PrSMs) Without: 10, with: 24 (3 PrSMs) Without: 10, with: 25 (3 PrSMs) Without: 10, with: 26 (2 PrSMs) Without: 10, with: 27 (1 PrSM) Without: 10, with: 28 (2 PrSMs) Without: 10, with: 29 (2 PrSMs) Without: 11, with: 12 (2 PrSMs) Without: 11, with: 13 (4 PrSMs) Without: 11, with: 14 (5 PrSMs) Without: 11, with: 15 (10 PrSMs) Without: 11, with: 16 (7 PrSMs) Without: 11, with: 17 (9 PrSMs) Without: 11, with: 18 (10 PrSMs) Without: 11, with: 19 (8 PrSMs) Without: 11, with: 20 (8 PrSMs) Without: 11, with: 21 (8 PrSMs) Without: 11, with: 22 (6 PrSMs) Without: 11, with: 23 (5 PrSMs) Without: 11, with: 24 (6 PrSMs) Without: 11, with: 25 (2 PrSMs) Without: 11, with: 26 (2 PrSMs) Without: 11, with: 27 (2 PrSMs) Without: 11, with: 29 (4 PrSMs) Without: 11, with: 31 (1 PrSM) Without: 12, with: 12 (1 PrSM) Without: 12, with: 14 (1 PrSM) Without: 12, with: 15 (5 PrSMs) Without: 12, with: 16 (5 PrSMs) Without: 12, with: 17 (8 PrSMs) Without: 12, with: 18 (8 PrSMs) Without: 12, with: 19 (11 PrSMs) Without: 12, with: 20 (5 PrSMs) Without: 12, with: 21 (5 PrSMs) Without: 12, with: 22 (8 PrSMs) Without: 12, with: 23 (6 PrSMs) Without: 12, with: 24 (3 PrSMs) Without: 12, with: 25 (8 PrSMs) Without: 12, with: 26 (3 PrSMs) Without: 12, with: 27 (1 PrSM) Without: 12, with: 28 (4 PrSMs) Without: 12, with: 29 (2 PrSMs) Without: 12, with: 31 (2 PrSMs) Without: 12, with: 32 (3 PrSMs) Without: 12, with: 33 (2 PrSMs) Without: 12, with: 34 (2 PrSMs) Without: 13, with: 15 (2 PrSMs) Without: 13, with: 16 (4 PrSMs) Without: 13, with: 17 (4 PrSMs) Without: 13, with: 18 (5 PrSMs) Without: 13, with: 19 (2 PrSMs) Without: 13, with: 20 (14 PrSMs) Without: 13, with: 21 (7 PrSMs) Without: 13, with: 22 (4 PrSMs) Without: 13, with: 23 (8 PrSMs) Without: 13, with: 24 (12 PrSMs) Without: 13, with: 25 (8 PrSMs) Without: 13, with: 26 (6 PrSMs) Without: 13, with: 27 (5 PrSMs) Without: 13, with: 28 (1 PrSM) Without: 13, with: 29 (5 PrSMs) Without: 13, with: 30 (2 PrSMs) Without: 13, with: 31 (3 PrSMs) Without: 13, with: 32 (3 PrSMs) Without: 13, with: 34 (1 PrSM) Without: 13, with: 35 (1 PrSM) Without: 13, with: 37 (1 PrSM) Without: 14, with: 15 (1 PrSM) Without: 14, with: 16 (1 PrSM) Without: 14, with: 17 (2 PrSMs) Without: 14, with: 18 (6 PrSMs) Without: 14, with: 19 (7 PrSMs) Without: 14, with: 20 (6 PrSMs) Without: 14, with: 21 (7 PrSMs) Without: 14, with: 22 (11 PrSMs) Without: 14, with: 23 (6 PrSMs) Without: 14, with: 24 (8 PrSMs) Without: 14, with: 25 (6 PrSMs) Without: 14, with: 26 (6 PrSMs) Without: 14, with: 27 (5 PrSMs) Without: 14, with: 28 (5 PrSMs) Without: 14, with: 29 (4 PrSMs) Without: 14, with: 30 (1 PrSM) Without: 14, with: 31 (5 PrSMs) Without: 14, with: 32 (2 PrSMs) Without: 14, with: 34 (3 PrSMs) Without: 14, with: 35 (1 PrSM) Without: 14, with: 36 (1 PrSM) Without: 14, with: 38 (1 PrSM) Without: 14, with: 41 (1 PrSM) Without: 15, with: 15 (1 PrSM) Without: 15, with: 16 (3 PrSMs) Without: 15, with: 17 (5 PrSMs) Without: 15, with: 18 (3 PrSMs) Without: 15, with: 19 (3 PrSMs) Without: 15, with: 20 (4 PrSMs) Without: 15, with: 21 (8 PrSMs) Without: 15, with: 22 (8 PrSMs) Without: 15, with: 23 (10 PrSMs) Without: 15, with: 24 (12 PrSMs) Without: 15, with: 25 (6 PrSMs) Without: 15, with: 26 (9 PrSMs) Without: 15, with: 27 (3 PrSMs) Without: 15, with: 28 (8 PrSMs) Without: 15, with: 29 (2 PrSMs) Without: 15, with: 30 (3 PrSMs) Without: 15, with: 31 (7 PrSMs) Without: 15, with: 32 (8 PrSMs) Without: 15, with: 33 (3 PrSMs) Without: 15, with: 34 (2 PrSMs) Without: 15, with: 35 (1 PrSM) Without: 15, with: 36 (3 PrSMs) Without: 15, with: 39 (1 PrSM) Without: 15, with: 40 (1 PrSM) Without: 16, with: 17 (1 PrSM) Without: 16, with: 18 (8 PrSMs) Without: 16, with: 19 (6 PrSMs) Without: 16, with: 20 (6 PrSMs) Without: 16, with: 21 (6 PrSMs) Without: 16, with: 22 (4 PrSMs) Without: 16, with: 23 (1 PrSM) Without: 16, with: 24 (6 PrSMs) Without: 16, with: 25 (12 PrSMs) Without: 16, with: 26 (14 PrSMs) Without: 16, with: 27 (6 PrSMs) Without: 16, with: 28 (6 PrSMs) Without: 16, with: 29 (9 PrSMs) Without: 16, with: 30 (4 PrSMs) Without: 16, with: 31 (3 PrSMs) Without: 16, with: 32 (4 PrSMs) Without: 16, with: 33 (3 PrSMs) Without: 16, with: 34 (3 PrSMs) Without: 16, with: 35 (1 PrSM) Without: 16, with: 37 (2 PrSMs) Without: 17, with: 18 (2 PrSMs) Without: 17, with: 19 (2 PrSMs) Without: 17, with: 20 (1 PrSM) Without: 17, with: 21 (2 PrSMs) Without: 17, with: 22 (4 PrSMs) Without: 17, with: 23 (9 PrSMs) Without: 17, with: 24 (7 PrSMs) Without: 17, with: 25 (11 PrSMs) Without: 17, with: 26 (9 PrSMs) Without: 17, with: 27 (5 PrSMs) Without: 17, with: 28 (9 PrSMs) Without: 17, with: 29 (9 PrSMs) Without: 17, with: 30 (6 PrSMs) Without: 17, with: 31 (3 PrSMs) Without: 17, with: 32 (12 PrSMs) Without: 17, with: 33 (11 PrSMs) Without: 17, with: 34 (4 PrSMs) Without: 17, with: 35 (1 PrSM) Without: 17, with: 36 (3 PrSMs) Without: 17, with: 37 (1 PrSM) Without: 17, with: 39 (2 PrSMs) Without: 17, with: 41 (1 PrSM) Without: 18, with: 19 (2 PrSMs) Without: 18, with: 22 (3 PrSMs) Without: 18, with: 23 (5 PrSMs) Without: 18, with: 24 (8 PrSMs) Without: 18, with: 25 (4 PrSMs) Without: 18, with: 26 (3 PrSMs) Without: 18, with: 27 (12 PrSMs) Without: 18, with: 28 (8 PrSMs) Without: 18, with: 29 (5 PrSMs) Without: 18, with: 30 (9 PrSMs) Without: 18, with: 31 (8 PrSMs) Without: 18, with: 32 (6 PrSMs) Without: 18, with: 33 (6 PrSMs) Without: 18, with: 34 (3 PrSMs) Without: 18, with: 35 (7 PrSMs) Without: 18, with: 37 (3 PrSMs) Without: 18, with: 38 (5 PrSMs) Without: 18, with: 39 (1 PrSM) Without: 18, with: 41 (1 PrSM) Without: 18, with: 42 (1 PrSM) Without: 19, with: 21 (1 PrSM) Without: 19, with: 22 (2 PrSMs) Without: 19, with: 23 (5 PrSMs) Without: 19, with: 24 (5 PrSMs) Without: 19, with: 25 (4 PrSMs) Without: 19, with: 26 (4 PrSMs) Without: 19, with: 27 (11 PrSMs) Without: 19, with: 28 (4 PrSMs) Without: 19, with: 29 (3 PrSMs) Without: 19, with: 30 (10 PrSMs) Without: 19, with: 31 (9 PrSMs) Without: 19, with: 32 (7 PrSMs) Without: 19, with: 33 (8 PrSMs) Without: 19, with: 34 (4 PrSMs) Without: 19, with: 35 (3 PrSMs) Without: 19, with: 36 (3 PrSMs) Without: 19, with: 37 (3 PrSMs) Without: 19, with: 44 (1 PrSM) Without: 19, with: 45 (2 PrSMs) Without: 20, with: 23 (1 PrSM) Without: 20, with: 24 (1 PrSM) Without: 20, with: 25 (3 PrSMs) Without: 20, with: 26 (4 PrSMs) Without: 20, with: 27 (4 PrSMs) Without: 20, with: 28 (2 PrSMs) Without: 20, with: 29 (2 PrSMs) Without: 20, with: 30 (9 PrSMs) Without: 20, with: 31 (7 PrSMs) Without: 20, with: 32 (9 PrSMs) Without: 20, with: 33 (10 PrSMs) Without: 20, with: 34 (7 PrSMs) Without: 20, with: 35 (12 PrSMs) Without: 20, with: 36 (4 PrSMs) Without: 20, with: 37 (6 PrSMs) Without: 20, with: 38 (4 PrSMs) Without: 20, with: 39 (6 PrSMs) Without: 20, with: 40 (1 PrSM) Without: 20, with: 41 (1 PrSM) Without: 21, with: 23 (1 PrSM) Without: 21, with: 24 (2 PrSMs) Without: 21, with: 25 (2 PrSMs) Without: 21, with: 26 (3 PrSMs) Without: 21, with: 27 (6 PrSMs) Without: 21, with: 28 (5 PrSMs) Without: 21, with: 29 (5 PrSMs) Without: 21, with: 30 (5 PrSMs) Without: 21, with: 31 (8 PrSMs) Without: 21, with: 32 (8 PrSMs) Without: 21, with: 33 (10 PrSMs) Without: 21, with: 34 (4 PrSMs) Without: 21, with: 35 (9 PrSMs) Without: 21, with: 36 (9 PrSMs) Without: 21, with: 37 (4 PrSMs) Without: 21, with: 38 (2 PrSMs) Without: 21, with: 39 (3 PrSMs) Without: 21, with: 40 (4 PrSMs) Without: 21, with: 41 (1 PrSM) Without: 21, with: 42 (2 PrSMs) Without: 21, with: 43 (1 PrSM) Without: 21, with: 47 (1 PrSM) Without: 22, with: 23 (1 PrSM) Without: 22, with: 27 (1 PrSM) Without: 22, with: 28 (2 PrSMs) Without: 22, with: 29 (9 PrSMs) Without: 22, with: 30 (6 PrSMs) Without: 22, with: 31 (5 PrSMs) Without: 22, with: 32 (6 PrSMs) Without: 22, with: 33 (5 PrSMs) Without: 22, with: 34 (9 PrSMs) Without: 22, with: 35 (6 PrSMs) Without: 22, with: 36 (6 PrSMs) Without: 22, with: 37 (4 PrSMs) Without: 22, with: 38 (4 PrSMs) Without: 22, with: 39 (6 PrSMs) Without: 22, with: 40 (6 PrSMs) Without: 22, with: 41 (3 PrSMs) Without: 22, with: 42 (4 PrSMs) Without: 22, with: 43 (3 PrSMs) Without: 22, with: 44 (1 PrSM) Without: 23, with: 28 (2 PrSMs) Without: 23, with: 29 (4 PrSMs) Without: 23, with: 30 (5 PrSMs) Without: 23, with: 31 (10 PrSMs) Without: 23, with: 32 (8 PrSMs) Without: 23, with: 33 (5 PrSMs) Without: 23, with: 34 (7 PrSMs) Without: 23, with: 35 (4 PrSMs) Without: 23, with: 36 (5 PrSMs) Without: 23, with: 37 (2 PrSMs) Without: 23, with: 38 (4 PrSMs) Without: 23, with: 39 (4 PrSMs) Without: 23, with: 40 (6 PrSMs) Without: 23, with: 41 (2 PrSMs) Without: 23, with: 43 (2 PrSMs) Without: 23, with: 44 (3 PrSMs) Without: 23, with: 48 (1 PrSM) Without: 23, with: 49 (1 PrSM) Without: 24, with: 27 (1 PrSM) Without: 24, with: 28 (1 PrSM) Without: 24, with: 29 (1 PrSM) Without: 24, with: 30 (3 PrSMs) Without: 24, with: 31 (6 PrSMs) Without: 24, with: 32 (3 PrSMs) Without: 24, with: 33 (10 PrSMs) Without: 24, with: 34 (6 PrSMs) Without: 24, with: 35 (6 PrSMs) Without: 24, with: 36 (5 PrSMs) Without: 24, with: 37 (12 PrSMs) Without: 24, with: 38 (5 PrSMs) Without: 24, with: 39 (3 PrSMs) Without: 24, with: 40 (7 PrSMs) Without: 24, with: 41 (2 PrSMs) Without: 24, with: 42 (2 PrSMs) Without: 24, with: 43 (7 PrSMs) Without: 24, with: 44 (3 PrSMs) Without: 24, with: 46 (1 PrSM) Without: 24, with: 49 (1 PrSM) Without: 24, with: 51 (1 PrSM) Without: 25, with: 29 (3 PrSMs) Without: 25, with: 30 (2 PrSMs) Without: 25, with: 31 (2 PrSMs) Without: 25, with: 32 (7 PrSMs) Without: 25, with: 33 (5 PrSMs) Without: 25, with: 34 (4 PrSMs) Without: 25, with: 35 (6 PrSMs) Without: 25, with: 36 (7 PrSMs) Without: 25, with: 37 (13 PrSMs) Without: 25, with: 38 (9 PrSMs) Without: 25, with: 39 (4 PrSMs) Without: 25, with: 40 (5 PrSMs) Without: 25, with: 41 (5 PrSMs) Without: 25, with: 42 (4 PrSMs) Without: 25, with: 43 (6 PrSMs) Without: 25, with: 44 (2 PrSMs) Without: 25, with: 45 (1 PrSM) Without: 25, with: 46 (3 PrSMs) Without: 25, with: 47 (2 PrSMs) Without: 25, with: 48 (2 PrSMs) Without: 25, with: 55 (1 PrSM) Without: 26, with: 28 (1 PrSM) Without: 26, with: 31 (2 PrSMs) Without: 26, with: 32 (7 PrSMs) Without: 26, with: 33 (6 PrSMs) Without: 26, with: 34 (2 PrSMs) Without: 26, with: 35 (4 PrSMs) Without: 26, with: 36 (5 PrSMs) Without: 26, with: 37 (8 PrSMs) Without: 26, with: 38 (3 PrSMs) Without: 26, with: 39 (4 PrSMs) Without: 26, with: 40 (3 PrSMs) Without: 26, with: 41 (6 PrSMs) Without: 26, with: 42 (4 PrSMs) Without: 26, with: 43 (2 PrSMs) Without: 26, with: 44 (3 PrSMs) Without: 26, with: 45 (4 PrSMs) Without: 26, with: 46 (1 PrSM) Without: 26, with: 47 (2 PrSMs) Without: 26, with: 48 (1 PrSM) Without: 26, with: 49 (1 PrSM) Without: 26, with: 50 (1 PrSM) Without: 27, with: 31 (1 PrSM) Without: 27, with: 32 (3 PrSMs) Without: 27, with: 33 (2 PrSMs) Without: 27, with: 34 (3 PrSMs) Without: 27, with: 35 (5 PrSMs) Without: 27, with: 36 (5 PrSMs) Without: 27, with: 37 (4 PrSMs) Without: 27, with: 38 (4 PrSMs) Without: 27, with: 39 (7 PrSMs) Without: 27, with: 40 (8 PrSMs) Without: 27, with: 41 (5 PrSMs) Without: 27, with: 42 (4 PrSMs) Without: 27, with: 43 (5 PrSMs) Without: 27, with: 44 (5 PrSMs) Without: 27, with: 45 (4 PrSMs) Without: 27, with: 46 (1 PrSM) Without: 27, with: 47 (3 PrSMs) Without: 27, with: 48 (1 PrSM) Without: 27, with: 49 (1 PrSM) Without: 27, with: 50 (1 PrSM) Without: 27, with: 54 (1 PrSM) Without: 27, with: 57 (1 PrSM) Without: 28, with: 31 (1 PrSM) Without: 28, with: 33 (1 PrSM) Without: 28, with: 34 (2 PrSMs) Without: 28, with: 35 (3 PrSMs) Without: 28, with: 36 (5 PrSMs) Without: 28, with: 37 (6 PrSMs) Without: 28, with: 38 (6 PrSMs) Without: 28, with: 39 (4 PrSMs) Without: 28, with: 40 (3 PrSMs) Without: 28, with: 41 (6 PrSMs) Without: 28, with: 43 (7 PrSMs) Without: 28, with: 44 (4 PrSMs) Without: 28, with: 45 (2 PrSMs) Without: 28, with: 46 (4 PrSMs) Without: 28, with: 47 (3 PrSMs) Without: 28, with: 48 (2 PrSMs) Without: 28, with: 49 (3 PrSMs) Without: 28, with: 51 (1 PrSM) Without: 28, with: 53 (1 PrSM) Without: 28, with: 57 (1 PrSM) Without: 29, with: 34 (1 PrSM) Without: 29, with: 35 (1 PrSM) Without: 29, with: 36 (2 PrSMs) Without: 29, with: 37 (7 PrSMs) Without: 29, with: 38 (4 PrSMs) Without: 29, with: 40 (8 PrSMs) Without: 29, with: 41 (7 PrSMs) Without: 29, with: 42 (8 PrSMs) Without: 29, with: 43 (4 PrSMs) Without: 29, with: 44 (4 PrSMs) Without: 29, with: 45 (2 PrSMs) Without: 29, with: 46 (2 PrSMs) Without: 29, with: 47 (3 PrSMs) Without: 29, with: 48 (2 PrSMs) Without: 29, with: 49 (4 PrSMs) Without: 29, with: 50 (1 PrSM) Without: 29, with: 51 (2 PrSMs) Without: 29, with: 52 (1 PrSM) Without: 29, with: 58 (1 PrSM) Without: 30, with: 35 (2 PrSMs) Without: 30, with: 36 (2 PrSMs) Without: 30, with: 37 (4 PrSMs) Without: 30, with: 38 (7 PrSMs) Without: 30, with: 39 (1 PrSM) Without: 30, with: 40 (6 PrSMs) Without: 30, with: 41 (7 PrSMs) Without: 30, with: 42 (3 PrSMs) Without: 30, with: 43 (4 PrSMs) Without: 30, with: 44 (6 PrSMs) Without: 30, with: 45 (4 PrSMs) Without: 30, with: 46 (5 PrSMs) Without: 30, with: 47 (5 PrSMs) Without: 30, with: 48 (3 PrSMs) Without: 30, with: 50 (1 PrSM) Without: 30, with: 51 (1 PrSM) Without: 30, with: 52 (1 PrSM) Without: 30, with: 53 (2 PrSMs) Without: 30, with: 55 (1 PrSM) Without: 30, with: 59 (1 PrSM) Without: 31, with: 34 (1 PrSM) Without: 31, with: 35 (1 PrSM) Without: 31, with: 36 (4 PrSMs) Without: 31, with: 37 (3 PrSMs) Without: 31, with: 38 (2 PrSMs) Without: 31, with: 39 (3 PrSMs) Without: 31, with: 40 (6 PrSMs) Without: 31, with: 41 (5 PrSMs) Without: 31, with: 42 (6 PrSMs) Without: 31, with: 43 (4 PrSMs) Without: 31, with: 44 (5 PrSMs) Without: 31, with: 45 (3 PrSMs) Without: 31, with: 46 (8 PrSMs) Without: 31, with: 47 (3 PrSMs) Without: 31, with: 48 (4 PrSMs) Without: 31, with: 49 (2 PrSMs) Without: 31, with: 50 (1 PrSM) Without: 31, with: 51 (5 PrSMs) Without: 31, with: 52 (2 PrSMs) Without: 31, with: 53 (2 PrSMs) Without: 31, with: 54 (1 PrSM) Without: 31, with: 55 (2 PrSMs) Without: 31, with: 56 (3 PrSMs) Without: 31, with: 59 (1 PrSM) Without: 32, with: 37 (1 PrSM) Without: 32, with: 39 (3 PrSMs) Without: 32, with: 41 (7 PrSMs) Without: 32, with: 42 (3 PrSMs) Without: 32, with: 43 (5 PrSMs) Without: 32, with: 44 (7 PrSMs) Without: 32, with: 45 (7 PrSMs) Without: 32, with: 46 (10 PrSMs) Without: 32, with: 47 (1 PrSM) Without: 32, with: 48 (2 PrSMs) Without: 32, with: 50 (1 PrSM) Without: 32, with: 51 (2 PrSMs) Without: 32, with: 52 (1 PrSM) Without: 32, with: 53 (1 PrSM) Without: 32, with: 54 (2 PrSMs) Without: 32, with: 55 (1 PrSM) Without: 32, with: 56 (2 PrSMs) Without: 33, with: 37 (1 PrSM) Without: 33, with: 38 (2 PrSMs) Without: 33, with: 39 (1 PrSM) Without: 33, with: 40 (4 PrSMs) Without: 33, with: 41 (3 PrSMs) Without: 33, with: 42 (4 PrSMs) Without: 33, with: 43 (4 PrSMs) Without: 33, with: 44 (5 PrSMs) Without: 33, with: 45 (3 PrSMs) Without: 33, with: 46 (4 PrSMs) Without: 33, with: 47 (4 PrSMs) Without: 33, with: 48 (2 PrSMs) Without: 33, with: 49 (1 PrSM) Without: 33, with: 51 (3 PrSMs) Without: 33, with: 52 (5 PrSMs) Without: 33, with: 53 (3 PrSMs) Without: 33, with: 54 (1 PrSM) Without: 34, with: 38 (1 PrSM) Without: 34, with: 41 (1 PrSM) Without: 34, with: 42 (2 PrSMs) Without: 34, with: 44 (4 PrSMs) Without: 34, with: 45 (7 PrSMs) Without: 34, with: 46 (8 PrSMs) Without: 34, with: 47 (5 PrSMs) Without: 34, with: 48 (4 PrSMs) Without: 34, with: 49 (5 PrSMs) Without: 34, with: 50 (1 PrSM) Without: 34, with: 51 (4 PrSMs) Without: 34, with: 52 (3 PrSMs) Without: 34, with: 55 (2 PrSMs) Without: 34, with: 56 (1 PrSM) Without: 34, with: 57 (2 PrSMs) Without: 34, with: 61 (1 PrSM) Without: 35, with: 41 (1 PrSM) Without: 35, with: 43 (2 PrSMs) Without: 35, with: 44 (6 PrSMs) Without: 35, with: 45 (1 PrSM) Without: 35, with: 46 (7 PrSMs) Without: 35, with: 47 (8 PrSMs) Without: 35, with: 48 (2 PrSMs) Without: 35, with: 49 (6 PrSMs) Without: 35, with: 50 (4 PrSMs) Without: 35, with: 51 (8 PrSMs) Without: 35, with: 55 (1 PrSM) Without: 35, with: 61 (2 PrSMs) Without: 35, with: 62 (2 PrSMs) Without: 36, with: 41 (3 PrSMs) Without: 36, with: 42 (2 PrSMs) Without: 36, with: 43 (2 PrSMs) Without: 36, with: 44 (1 PrSM) Without: 36, with: 45 (2 PrSMs) Without: 36, with: 47 (1 PrSM) Without: 36, with: 48 (2 PrSMs) Without: 36, with: 49 (4 PrSMs) Without: 36, with: 50 (6 PrSMs) Without: 36, with: 51 (2 PrSMs) Without: 36, with: 52 (2 PrSMs) Without: 36, with: 53 (4 PrSMs) Without: 36, with: 54 (1 PrSM) Without: 37, with: 45 (3 PrSMs) Without: 37, with: 46 (1 PrSM) Without: 37, with: 47 (1 PrSM) Without: 37, with: 50 (4 PrSMs) Without: 37, with: 51 (3 PrSMs) Without: 37, with: 52 (3 PrSMs) Without: 37, with: 53 (1 PrSM) Without: 37, with: 54 (6 PrSMs) Without: 37, with: 55 (2 PrSMs) Without: 37, with: 56 (1 PrSM) Without: 37, with: 60 (1 PrSM) Without: 37, with: 61 (1 PrSM) Without: 37, with: 62 (1 PrSM) Without: 38, with: 45 (1 PrSM) Without: 38, with: 46 (2 PrSMs) Without: 38, with: 47 (2 PrSMs) Without: 38, with: 48 (1 PrSM) Without: 38, with: 49 (2 PrSMs) Without: 38, with: 51 (2 PrSMs) Without: 38, with: 52 (3 PrSMs) Without: 38, with: 53 (2 PrSMs) Without: 38, with: 55 (1 PrSM) Without: 38, with: 56 (3 PrSMs) Without: 38, with: 57 (2 PrSMs) Without: 38, with: 58 (1 PrSM) Without: 38, with: 59 (1 PrSM) Without: 38, with: 61 (2 PrSMs) Without: 38, with: 62 (1 PrSM) Without: 38, with: 63 (1 PrSM) Without: 39, with: 44 (2 PrSMs) Without: 39, with: 45 (2 PrSMs) Without: 39, with: 48 (2 PrSMs) Without: 39, with: 49 (5 PrSMs) Without: 39, with: 51 (2 PrSMs) Without: 39, with: 52 (4 PrSMs) Without: 39, with: 53 (3 PrSMs) Without: 39, with: 54 (4 PrSMs) Without: 39, with: 55 (2 PrSMs) Without: 39, with: 56 (3 PrSMs) Without: 39, with: 58 (2 PrSMs) Without: 39, with: 59 (1 PrSM) Without: 39, with: 60 (2 PrSMs) Without: 40, with: 44 (1 PrSM) Without: 40, with: 45 (2 PrSMs) Without: 40, with: 50 (2 PrSMs) Without: 40, with: 51 (2 PrSMs) Without: 40, with: 52 (5 PrSMs) Without: 40, with: 53 (3 PrSMs) Without: 40, with: 54 (3 PrSMs) Without: 40, with: 55 (2 PrSMs) Without: 40, with: 56 (2 PrSMs) Without: 40, with: 57 (1 PrSM) Without: 40, with: 58 (1 PrSM) Without: 40, with: 59 (1 PrSM) Without: 40, with: 60 (1 PrSM) Without: 40, with: 61 (1 PrSM) Without: 40, with: 62 (1 PrSM) Without: 40, with: 63 (2 PrSMs) Without: 40, with: 68 (1 PrSM) Without: 41, with: 48 (1 PrSM) Without: 41, with: 49 (2 PrSMs) Without: 41, with: 50 (3 PrSMs) Without: 41, with: 51 (3 PrSMs) Without: 41, with: 52 (1 PrSM) Without: 41, with: 53 (1 PrSM) Without: 41, with: 54 (2 PrSMs) Without: 41, with: 55 (2 PrSMs) Without: 41, with: 56 (2 PrSMs) Without: 41, with: 57 (1 PrSM) Without: 41, with: 59 (3 PrSMs) Without: 41, with: 60 (2 PrSMs) Without: 41, with: 61 (1 PrSM) Without: 41, with: 63 (1 PrSM) Without: 41, with: 69 (1 PrSM) Without: 42, with: 50 (1 PrSM) Without: 42, with: 51 (1 PrSM) Without: 42, with: 52 (1 PrSM) Without: 42, with: 53 (2 PrSMs) Without: 42, with: 54 (1 PrSM) Without: 42, with: 55 (3 PrSMs) Without: 42, with: 56 (2 PrSMs) Without: 42, with: 57 (2 PrSMs) Without: 42, with: 58 (2 PrSMs) Without: 42, with: 59 (1 PrSM) Without: 42, with: 60 (2 PrSMs) Without: 42, with: 61 (2 PrSMs) Without: 42, with: 64 (1 PrSM) Without: 42, with: 66 (1 PrSM) Without: 42, with: 67 (1 PrSM) Without: 42, with: 79 (1 PrSM) Without: 43, with: 46 (1 PrSM) Without: 43, with: 48 (1 PrSM) Without: 43, with: 49 (1 PrSM) Without: 43, with: 51 (1 PrSM) Without: 43, with: 52 (2 PrSMs) Without: 43, with: 53 (3 PrSMs) Without: 43, with: 54 (5 PrSMs) Without: 43, with: 56 (3 PrSMs) Without: 43, with: 57 (1 PrSM) Without: 43, with: 58 (4 PrSMs) Without: 43, with: 60 (2 PrSMs) Without: 43, with: 61 (1 PrSM) Without: 43, with: 63 (3 PrSMs) Without: 43, with: 64 (3 PrSMs) Without: 43, with: 68 (1 PrSM) Without: 44, with: 51 (1 PrSM) Without: 44, with: 52 (3 PrSMs) Without: 44, with: 54 (3 PrSMs) Without: 44, with: 55 (2 PrSMs) Without: 44, with: 56 (1 PrSM) Without: 44, with: 57 (1 PrSM) Without: 44, with: 58 (4 PrSMs) Without: 44, with: 59 (4 PrSMs) Without: 44, with: 61 (1 PrSM) Without: 44, with: 62 (2 PrSMs) Without: 44, with: 63 (2 PrSMs) Without: 44, with: 64 (1 PrSM) Without: 44, with: 65 (1 PrSM) Without: 44, with: 66 (1 PrSM) Without: 44, with: 68 (3 PrSMs) Without: 44, with: 71 (1 PrSM) Without: 45, with: 52 (2 PrSMs) Without: 45, with: 55 (2 PrSMs) Without: 45, with: 56 (2 PrSMs) Without: 45, with: 57 (2 PrSMs) Without: 45, with: 58 (2 PrSMs) Without: 45, with: 59 (1 PrSM) Without: 45, with: 60 (1 PrSM) Without: 45, with: 61 (1 PrSM) Without: 45, with: 62 (2 PrSMs) Without: 45, with: 63 (1 PrSM) Without: 45, with: 64 (1 PrSM) Without: 45, with: 65 (2 PrSMs) Without: 45, with: 66 (2 PrSMs) Without: 45, with: 69 (1 PrSM) Without: 46, with: 52 (1 PrSM) Without: 46, with: 55 (2 PrSMs) Without: 46, with: 56 (1 PrSM) Without: 46, with: 57 (3 PrSMs) Without: 46, with: 58 (3 PrSMs) Without: 46, with: 61 (2 PrSMs) Without: 46, with: 65 (1 PrSM) Without: 46, with: 66 (1 PrSM) Without: 46, with: 67 (1 PrSM) Without: 46, with: 77 (1 PrSM) Without: 46, with: 82 (1 PrSM) Without: 47, with: 53 (2 PrSMs) Without: 47, with: 57 (3 PrSMs) Without: 47, with: 58 (2 PrSMs) Without: 47, with: 59 (2 PrSMs) Without: 47, with: 60 (1 PrSM) Without: 47, with: 61 (1 PrSM) Without: 47, with: 62 (1 PrSM) Without: 47, with: 63 (2 PrSMs) Without: 47, with: 64 (2 PrSMs) Without: 47, with: 65 (1 PrSM) Without: 47, with: 66 (1 PrSM) Without: 47, with: 67 (1 PrSM) Without: 47, with: 68 (1 PrSM) Without: 47, with: 69 (2 PrSMs) Without: 47, with: 70 (2 PrSMs) Without: 47, with: 77 (1 PrSM) Without: 47, with: 78 (1 PrSM) Without: 48, with: 55 (1 PrSM) Without: 48, with: 56 (1 PrSM) Without: 48, with: 57 (1 PrSM) Without: 48, with: 59 (1 PrSM) Without: 48, with: 60 (1 PrSM) Without: 48, with: 61 (1 PrSM) Without: 48, with: 62 (1 PrSM) Without: 48, with: 63 (3 PrSMs) Without: 48, with: 64 (2 PrSMs) Without: 48, with: 65 (1 PrSM) Without: 48, with: 72 (1 PrSM) Without: 48, with: 77 (1 PrSM) Without: 49, with: 57 (1 PrSM) Without: 49, with: 58 (1 PrSM) Without: 49, with: 61 (4 PrSMs) Without: 49, with: 62 (1 PrSM) Without: 49, with: 63 (2 PrSMs) Without: 49, with: 64 (3 PrSMs) Without: 49, with: 67 (2 PrSMs) Without: 49, with: 69 (1 PrSM) Without: 49, with: 73 (1 PrSM) Without: 49, with: 80 (1 PrSM) Without: 50, with: 58 (1 PrSM) Without: 50, with: 62 (2 PrSMs) Without: 50, with: 64 (4 PrSMs) Without: 50, with: 65 (1 PrSM) Without: 50, with: 66 (1 PrSM) Without: 50, with: 67 (1 PrSM) Without: 50, with: 79 (1 PrSM) Without: 51, with: 61 (1 PrSM) Without: 51, with: 64 (1 PrSM) Without: 51, with: 65 (1 PrSM) Without: 51, with: 66 (1 PrSM) Without: 51, with: 67 (2 PrSMs) Without: 51, with: 68 (1 PrSM) Without: 51, with: 69 (1 PrSM) Without: 51, with: 74 (1 PrSM) Without: 51, with: 78 (1 PrSM) Without: 51, with: 91 (1 PrSM) Without: 52, with: 59 (1 PrSM) Without: 52, with: 61 (1 PrSM) Without: 52, with: 64 (3 PrSMs) Without: 52, with: 65 (1 PrSM) Without: 52, with: 66 (1 PrSM) Without: 52, with: 69 (1 PrSM) Without: 52, with: 70 (1 PrSM) Without: 52, with: 72 (1 PrSM) Without: 53, with: 59 (1 PrSM) Without: 53, with: 64 (1 PrSM) Without: 53, with: 65 (2 PrSMs) Without: 53, with: 66 (1 PrSM) Without: 53, with: 67 (1 PrSM) Without: 53, with: 69 (2 PrSMs) Without: 53, with: 72 (1 PrSM) Without: 53, with: 73 (1 PrSM) Without: 53, with: 75 (2 PrSMs) Without: 54, with: 58 (1 PrSM) Without: 54, with: 64 (1 PrSM) Without: 54, with: 65 (1 PrSM) Without: 54, with: 66 (1 PrSM) Without: 54, with: 67 (1 PrSM) Without: 54, with: 70 (1 PrSM) Without: 54, with: 72 (1 PrSM) Without: 54, with: 75 (1 PrSM) Without: 55, with: 62 (1 PrSM) Without: 55, with: 66 (1 PrSM) Without: 55, with: 70 (1 PrSM) Without: 55, with: 71 (1 PrSM) Without: 55, with: 73 (1 PrSM) Without: 55, with: 77 (1 PrSM) Without: 56, with: 64 (1 PrSM) Without: 56, with: 67 (1 PrSM) Without: 56, with: 68 (2 PrSMs) Without: 56, with: 72 (1 PrSM) Without: 56, with: 78 (1 PrSM) Without: 57, with: 67 (1 PrSM) Without: 57, with: 70 (1 PrSM) Without: 57, with: 72 (1 PrSM) Without: 57, with: 77 (1 PrSM) Without: 57, with: 81 (1 PrSM) Without: 57, with: 83 (1 PrSM) Without: 58, with: 64 (1 PrSM) Without: 58, with: 70 (2 PrSMs) Without: 58, with: 78 (1 PrSM) Without: 59, with: 72 (1 PrSM) Without: 59, with: 78 (2 PrSMs) Without: 59, with: 80 (1 PrSM) Without: 59, with: 85 (1 PrSM) Without: 60, with: 67 (1 PrSM) Without: 60, with: 74 (1 PrSM) Without: 60, with: 80 (1 PrSM) Without: 60, with: 86 (1 PrSM) Without: 61, with: 71 (1 PrSM) Without: 61, with: 72 (1 PrSM) Without: 61, with: 79 (1 PrSM) Without: 61, with: 85 (1 PrSM) Without: 62, with: 77 (1 PrSM) Without: 62, with: 84 (1 PrSM) Without: 63, with: 72 (2 PrSMs) Without: 63, with: 75 (1 PrSM) Without: 63, with: 87 (1 PrSM) Without: 64, with: 73 (2 PrSMs) Without: 64, with: 74 (1 PrSM) Without: 65, with: 75 (1 PrSM) Without: 65, with: 77 (1 PrSM) Without: 65, with: 81 (1 PrSM) Without: 65, with: 92 (1 PrSM) Without: 66, with: 86 (1 PrSM) Without: 67, with: 75 (1 PrSM) Without: 67, with: 78 (1 PrSM) Without: 67, with: 84 (1 PrSM) Without: 68, with: 82 (1 PrSM) Without: 69, with: 80 (1 PrSM) Without: 69, with: 85 (1 PrSM) Without: 69, with: 103 (1 PrSM) Without: 70, with: 86 (1 PrSM) Without: 70, with: 87 (1 PrSM) Without: 71, with: 92 (1 PrSM) Without: 72, with: 85 (1 PrSM) Without: 75, with: 94 (1 PrSM) Without: 76, with: 88 (1 PrSM) Without: 78, with: 100 (1 PrSM) Without: 80, with: 102 (1 PrSM) Without: 81, with: 99 (1 PrSM) Without: 83, with: 103 (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 spectrum102114
Matched fragment masses2234
Matched fragment ions1830

Post mass matching added a median of 12 matched fragment masses per PrSM (interquartile range 8 to 15, maximum 40). The number of matched fragment ions per PrSM rose from a median of 18 to 30. The added masses are mostly small (median 1.27 kDa) and carry low charges (77% are singly or doubly charged).