Electronic Supporting Information
Proteome characterization of the unsequenced psychrophile
Pedobacter cryoconitis using 15N metabolic labeling, tandem
mass spectrometry and a new bioinformatic workflow.
Ana G. Pereira-Medrano,† Rosa Margesin,‡ Phillip C. Wright†,*
† Biological and Environmental Systems Group, ChELSI Institute, Chemical and Process Engineering,
The University of Sheffield, Mappin Street, Sheffield, S1 3JD, and ‡ Institut für Mikrobiologie,
Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria
EXPERIMENTAL PROCEDURE
2-DE
The extracts were mixed with Destreak Rehydration solution (Amersham Bioscience, Little Chalfont,
UK). IPG strips (3-10 pH) were passively rehydrated overnight. 300 µg (workflows A and B) and 1,000
µg (workflow faster B) of total proteins (Table 1) were cup-loaded and IEF was performed using a threestep protocol with a Protean IEF cell (BioRad), as detailed elsewhere.1 Focused strips were incubated for
15 min in a solution containing 6 M urea, 2% SDS, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, and 2%
(w/v) DTT, then 15 min in a solution containing 6 M urea, 2% SDS, 0.375 M Tris-HCl (pH 8.8), 20%
glycerol, and 4% Iodoacetamide. SDS PAGE was then performed in 12%T (12% acrylamide + 0.32%
N,N-methylenbisacrylamide), 2.6%C (% N,N methylenbisacrylamide 100/T) slab gel (17 cm x 17 cm x 1
mm). Electrophoresis was carried out with two steps of constant current: 16 mAgel-1 for 30 min and 24
mAgel-1 for approximately 5 hours.
Workflow A - Ortholog database search with manual validation
In workflow A (Figure 1) MS/MS data from each spot were analysed using Mascot, as detailed above,
followed by manual quantitation. The ortholog search was carried out twice for each MS/MS spectrum:
(1) 14N monoisotopic mass values of the 20 amino acids; (2) with the calculated amino acids mass values
using
15
N. Peptide identifications from the first search were from the light (unlabelled) version of the
proteins, and identifications from the second search were from the heavy (labelled) version. In parallel,
ortholog protein identification was carried out with Peaks Studio 4.5. The same concatenated database
and search parameters were used, as for Mascot. When using Peaks Protein ID in Peaks Studio 4.5
(Bioinformatics Solution, ON, Canada)2 for an ortholog search, a confident protein identification had ≥2
unique peptides with Peaks peptide scores ≥50, as recommended by Peaks developers (written
communication, 28 August 2007). Also, the same two ortholog searches ( 14N then 15N) were carried out
in Peaks for each MS/MS spectrum. Peptides with the two versions (light or unlabelled, and heavy or
1
labelled) were validated by the N-constraint. If no validated protein identifications were obtained for the
spot, a similarity database search was then performed, as indicated below. Manual quantitation was
carried out for the validated protein identifications, based on a previous report. 1 First, manual inspection
of each peptide spectrum was performed to confirm the presence of the light and heavy versions of each
peptide. The mass difference of the monoisotopic peaks between both peptide versions was calculated
and compared to the number of nitrogen atoms present in the identified peptide to confirm that the peaks
corresponded
to
the
same
peptide,
using
an
online
Proteomics
Toolkit
(http://db.systemsbiology.net:8080/proteomicsToolkit/IsotopeServlet.html). Peptide peak areas of both
versions in the TOF-MS scans were integrated over time using the LC-MS reconstruct tool in the Analyst
Software package (Applied Biosystems, Foster City, CA, USA). The relative quantitation of each peptide
was calculated by dividing the peptide peak areas of the heavy version with the light version. The
average 15N:14N ratio of all the peptides from a homologous protein and their coefficient of variation was
then calculated. The CV of the ratios of the biological replicates was also calculated.
Workflow A - Similarity database search using MS Blast
MS/MS data from spots without valid ortholog protein identification (workflow A Figure 1) were
analysed by a similarity database search. Each spectrum was interpreted by Peaks to calculate de novo
peptide sequences, using the same concatenated database and search parameters described above for
Mascot ortholog searches. The de novo prediction of peptide sequences was carried out twice for each
tandem MS spectrum, once with calculated 14N amino acids mass values, secondly with 15N mass values.
Only de novo peptide sequences that resulted in the light (14N) and heavy (15N) versions, of 6 or more
amino acids, and with a 80% Peaks de novo sequencing score were selected for further analysis. MS
BLAST was used for protein identification of the selected peptide sequences (http://dove.emblheidelberg.de/Blast2/msblast.html).3 The search was performed against the on-line updated entire nonredundant database at the NCBI (June to October 2008). Confident protein identifications from a
similarity database searches considered only proteins with MS BLAST with ≥2 peptides with high
scoring pairs (HSPs – a region of high local sequence similarity between the peptide in the query and the
protein in a database that was identified by database searching), and with a statistically confident protein
hit according to the MS BLAST scoring scheme (ie, a total BLAST score of ≥100).4 When using Peaks
Studio 4.5 for de novo sequencing and similarity searches with Spider, only proteins with ≥2 unique
peptide, with >79% Peaks de novo sequencing score, and peptide RSD (Relative Sequence Distance)
values <0.5 were deemed confident protein identifications as indicated by the developers (written
communication, 14 May 2008). The RSD (Relative Sequence Distance) values5 were used to select
confident peptide identifications instead of the Spider scores. As reported by the developers, the Spider
score calculated in Peaks Studio 4.5 is a measurement of the degree of homology from the database
sequences to the experimental peptide by using the partially correct sequence tag, rather than a
probability of a match being correct. Hence, only de novo peptide sequences with a high score (>79%)
2
and with an RSD value <0.5 were selected as confident peptide identifications. Spider scores are not
reported in the protein lists. An RSD of 0 means that the de novo sequence is identical to the true peptide
sequence, and one means that the de novo sequence is completely different from the true peptide
sequence. In both similarity database searches, de novo peptide sequences with a score Peaks de novo
>79% were selected, a threshold that allowed for more flexibility to search for low abundance proteins,
but is not as stringent as another study reported (selected only 100% confident sequences).6 However, it
is still in a high confidence score range,2 and was further validated using the N-constraint in any case.
100
100
80
80
60
60
40
40
20
20
0
Rel. growth (OD600, %)
Rel. protease production
(%)
Supporting Figure 1. Number of publications per year on proteomic studies of cold adaptation,
according to PubMed (up to December 2010). Insert shows the taxonomy of organisms reported in all
these publications. From all these publications, only 8 psychrophiles (microorganisms that adapt to cold
environments,) have been studied.
0
1
5
10
15
20
25
Temperature (°C)
Protease production
Growth
Supporting Figure 1. Effect of temperature on growth and protease production by P. cryoconitis after 72
h of cultivation (modified after {Margesin, 2005 #391})
3
Supporting Figure 2. 2-DE gel image of aligned gel of two biological replicates of Pedobacter
cryoconitis protein sample from the glucose mix (Table 1) stained with Coomassie Brilliant Blue G-250.
The polypeptide molecular mass scale in kDa is depicted on the y-axis while the x-axis shows the pI
range. The proteins were resolved in 3–10 linear pH gradient (IPG strip) and 12% SDS-PAGE (1700 ×
1700 × 1 mm). Arrows represent spots that were excised, based on the spots with the largest normalised
volume (calculated using Progenesis SameSpots v2.0), and numbered 1 to 120 from the larges to the
smallest. Normalised volume values are detailed in Supporting Table 1. These excised spots were in-gel
digested with trypsin, identified by MS and results of the proteins are shown in Supporting Table 3A,
corresponding to the same numbers.
4
Supporting Figure 3. 2-DE gel image of aligned gel of two biological replicates of Pedobacter
cryoconitis protein sample from the maltose mix (Table 1) stained with Coomassie Brilliant Blue G-250.
The polypeptide molecular mass scale in kDa is depicted on the y-axis while the x-axis shows the pI
range. The proteins were resolved in 3–10 linear pH gradient (IPG strip) and 12% SDS-PAGE (1700 ×
1700 × 1 mm). Arrows represent spots that were excised, based on the spots with the largest normalised
volume (calculated using Progenesis SameSpots v2.0), and numbered 1 to 120 from the larges to the
smallest. Normalised volume values are detailed in Supporting Table 1. These excised spots were in-gel
digested with trypsin, identified by MS and results of the proteins are shown in Supporting Table 3B,
corresponding to the same numbers.
5
Supporting Table 1. List of 120 spots with highest normalized volumes from the aligned 2-DE gel
image from the glucose and maltose mix (each gel for a biological replicate), selected for identification
and quantitation. Only spots that appeared in both gels were selected, hence an aligned gel was needed
(no significant fold change, <1.5). Alignment as well as the volume and normalized volume
measurements of two 2-DE gels was carried out with Progenesis SameSpots v2.0 (Nonlinear Dynamics,
Newcastle, UK). The same 120 spots were excised for each of the two gels for MS/MS analysis, protein
identification and quantitation.
Spot number
in order of
volume
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
Glucose Mix
Volume (CV)
7,896,623.0 (1%)
5,026,072.5 (4%)
4,699,548.0 (9%)
4,676,346.5 (2%)
3,978,665.0 (4%)
3,858,627.5 (11%)
3,697,712.5 (0%)
3,653,610.0 (7%)
3,368,045.5 (19%)
3,241,776.0 (11%)
3,064,420.0 (23%)
2,741,511.5 (18%)
2,702,461.5 (1%)
2,573,535.0 (5%)
2,523,894.0 (3%)
2,519,510.5 (3%)
2,482,973.0 (0%)
2,427,556.5 (12%)
2,423,479.5 (29%)
2,374,486.0 (2%)
2,367,701.0 (2%)
2,311,027.5 (6%)
2,262,825.0 (9%)
2,259,619.0 (10%)
2,251,352.5 (3%)
2,226,436.5 (11%)
2,214,736.0 (26%)
2,042,975.5 (10%)
1,939,727.5 (11%)
1,936,053.0 (5%)
1,856,040.0 (29%)
1,689,928.0 (21%)
1,661,617.5 (22%)
1,642,726.0 (25%)
1,551,500.5 (10%)
1,538,377.5 (12%)
1,494,811.0 (7%)
1,468,856.5 (31%)
1,442,867.0 (26%)
1,397,794.5 (1%)
1,384,934.0 (11%)
1,356,203.0 (10%)
1,332,565.0 (7%)
1,322,049.5 (22%)
1,312,297.0 (12%)
1,278,820.5 (12%)
1,262,084.0 (42%)
1,251,816.0 (22%)
1,184,584.0 (12%)
1,181,348.5 (10%)
1,181,061.0 (12%)
1,179,329.5 (14%)
1,175,624.5 (7%)
1,166,639.5 (8%)
1,141,433.5 (5%)
Maltose Mix
Normalised Volume (CV)
20,383,545 (3%)
10,262,929 (2%)
16,438.781 (4%)
12,966.024 (1%)
12,144.219 (12%)
12,073.480 (5%)
9,973.781 (13%)
9,543.468 (2%)
9,420.909 (4%)
8,670.233 (17%)
8,379.517 (14%)
7,885.138 (20%)
7,058.794 (15%)
6,973.975 (1%)
6,646.750 (8%)
6,516.771 (6%)
6,500.684 (0%)
6,408.914 (3%)
6,255.688 (9%)
6,230.673 (26%)
6,130.091 (4%)
5,959.890 (3%)
5,840.295 (13%)
5,833.374 (6%)
5,808.773 (0%)
5,755.282 (14%)
5,695.819 (24%)
5,280.432 (13%)
5,014.227 (14%)
4,993.306 (3%)
4,809.257 (32%)
4,373.926 (23%)
4,275.749 (20%)
4,254.145 (27%)
3,999.138 (7%)
3,977.229 (15%)
3,854.677 (4%)
3,775.325 (28%)
3,710.808 (24%)
3,608.239 (4%)
3,579.768 (13%)
3,505.226 (13%)
3,435.895 (5%)
3,422.261 (24%)
3,392.358 (14%)
3,295.244 (9%)
3,240.377 (24%)
3,236.444 (39%)
3,053.269 (13%)
3,053.251 (15%)
3,052.618 (9%)
3,037.930 (12%)
3,037.372 (10%)
3,014.414 (11%)
2,944.171 (2%)
Volume (CV)
6,876,520.0 (19%)
5,906,462.5 (14%)
5,381,578.5 (16%)
4,501,478.5 (2%)
4,286,703.5 (8%)
4,197,421.0 (1%)
3,430,525.0 (12%)
3,198,604.5 (5%)
3,120,757.5 (21%)
3,088,817.5 (22%)
2,698,294.0 (40%)
2,557,327.0 (52%)
2,384,235.0 (15%)
2,328,700.0 (11%)
2,109,313.5 (51%)
2,082,116.0 (15%)
2,075,511.5 (10%)
2,069,926.5 (2%)
2,015,265.0 (5%)
1,969,187.5 (2%)
1,899,705.0 (0%)
1,837,351.0 (27%)
1,798,798.5 (8%)
1,771,484.0 (7%)
1,766,717.5 (4%)
1,757,258.5 (5%)
1,729,747.0 (30%)
1,687,488.0 (7%)
1,677,568.0 (77%)
1,670,988.0 (97%)
1,608,803.0 (24%)
1,605,314.0 (45%)
1,597,815.0 (68%)
1,587,272.5 (79%)
1,586,384.5 (12%)
1,567,205.5 (9%)
1,560,155.5 (57%)
1,524,571.5 (37%)
1,523,405.5 (6%)
1,498,284.0 (8%)
1,422,649.5 (20%)
1,402,059.0 (60%)
1,378,250.0 (92%)
1,324,773.5 (20%)
1,318,161.0 (45%)
1,310,008.5 (41%)
1,259,948.0 (7%)
1,257,099.5 (10%)
1,212,577.0 (1%)
1,190,815.0 (2%)
1,158,536.0 (14%)
1,141,754.5 (1%)
1,129,614.0 (3%)
1,123,600.0 (60%)
1,117,172.5 (30%)
6
Normalised Volume (CV)
21,148.015 (34%)
17,708.104 (2%)
16,105.691 (1%)
13,619.882 (13%)
12,915.133 (8%)
12,717.627 (15%)
10,303.189 (4%)
9,732.364 (20%)
9,303.990 (6%)
9,204.599 (6%)
7,924.827 (25%)
7,440.125 (38%)
7,142.211 (1%)
6,996.014 (4%)
6,264.040 (14%)
6,241.033 (5%)
6,236.919 (0%)
6,138.881 (37%)
6,130.971 (20%)
5,958.187 (13%)
5,756.026 (15%)
5,484.323 (23%)
5,452.381 (12%)
5,397.198 (22%)
5,338.599 (12%)
5,306.547 (11%)
5,118.688 (15%)
5,086.937 (9%)
4,786.249 (8%)
4,780.285 (65%)
4,764.061 (4%)
4,715.704 (6%)
4,694.550 (31%)
4,684.010 (88%)
4,640.508 (22%)
4,587.176 (55%)
4,519.203 (43%)
4,514.253 (68%)
4,512.227 (7%)
4,488.144 (22%)
4,379.635 (35%)
4,096.122 (54%)
4,051.540 (47%)
3,952.435 (5%)
3,878.264 (82%)
3,857.000 (30%)
3,838.933 (25%)
3,798.331 (9%)
3,677.664 (16%)
3,615.888 (18%)
3,473.581 (2%)
3,458.491 (15%)
3,431.740 (18%)
3,346.564 (19%)
3,333.675 (6%)
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
1,137,739.5 (5%)
1,104,209.0 (5%)
1,103,579.5 (6%)
1,097,326.5 (23%)
1,090,336.0 (6%)
1,088,969.5 (2%)
1,086,827.0 (17%)
1,047,714.5 (8%)
1,035,372.5 (43%)
1,031,846.0 (6%)
997,979.5 (14%)
994,360.5 (18%)
980,571.5 (1%)
973,738.0 (2%)
967,367.5 (14%)
966,933.0 (20%)
963,137.5 (17%)
952,701.0 (37%)
941,069.0 (1%)
938,158.0 (8%)
925,674.5 (22%)
912,334.0 (1%)
911,343.5 (12%)
908,171.5 (53%)
908,075.0 (4%)
888,555.5 (12%)
888,263.5 (30%)
870,555.0 (7%)
863,586.5 (8%)
860,737.0 (21%)
846,642.0 (4%)
835,317.5 (5%)
826,423.0 (10%)
778,999.0 (13%)
771,180.0 (14%)
766,324.0 (3%)
739,911.5 (6%)
736,293.0 (4%)
685,413.5 (8%)
680,887.0 (3%)
671,134.0 (5%)
665,163.5 (20%)
658,095.5 (13%)
641,372.5 (1%)
585,241.5 (5%)
580,266.0 (2%)
570,168.0 (1%)
563,650.5 (13%)
563,017.0 (46%)
542,348.5 (12%)
536,862.0 (12%)
530,313.5 (19%)
522,023.5 (4%)
520,301.5 (8%)
493,942.0 (6%)
492,259.0 (20%)
490,818.5 (7%)
483,456.0 (0%)
482,449.0 (6%)
479,319.0 (35%)
444,787.5 (16%)
386,611.0 (24%)
380,022.5 (30%)
366,449.5 (5%)
293,411.5 (25%)
2,934.690 (2%)
2,851.996 (8%)
2,846.014 (3%)
2,841.046 (26%)
2,816.428 (9%)
2,811.372 (5%)
2,798.817 (14%)
2,701.271 (5%)
2,661.108 (3%)
2,656.879 (40%)
2,570.908 (11%)
2,560.055 (16%)
2,530.468 (2%)
2,514.033 (5%)
2,501.460 (16%)
2,491.628 (20%)
2,488.872 (17%)
2,471.396 (40%)
2,429.292 (4%)
2,418.632 (6%)
2,382.167 (19%)
2,360.978 (56%)
2,354.373 (2%)
2,348.476 (9%)
2,344.887 (6%)
2,289.676 (9%)
2,283.192 (28%)
2,244.827 (4%)
2,231.408 (11%)
2,227.867 (24%)
2,186.253 (6%)
2,154.509 (2%)
2,135.987 (13%)
2,007.126 (10%)
1,994.240 (17%)
1,978.658 (6%)
1,911.394 (9%)
1,899.388 (1%)
1,770.963 (11%)
1,758.062 (6%)
1,731.049 (2%)
1,712.153 (17%)
1,701.476 (15%)
1,655.653 (4%)
1,511.494 (7%)
1,498.130 (5%)
1,471.778 (4%)
1,462.158 (48%)
1,452.330 (10%)
1,402.143 (15%)
1,383.415 (9%)
1,365.318 (16%)
1,348.007 (6%)
1,344.454 (11%)
1,273.756 (4%)
1,268.015 (10%)
1,267.027 (18%)
1,247.752 (2%)
1,246.303 (9%)
1,243.004 (38%)
1,150.503 (19%)
1,001.115 (27%)
984.891 (33%)
946.506 (8%)
759.829 (27%)
1,113,062.0 (21%)
1,108,296.0 (82%)
1,107,854.5 (9%)
1,101,364.0 (3%)
1,092,287.0 (2%)
1,034,156.0 (61%)
1,015,298.0 (34%)
1,014,824.5 (60%)
967,395.0 (90%)
964,607.0 (3%)
953,244.5 (16%)
945,018.5 (54%)
918,189.5 (0%)
887,702.5 (7%)
887,618.5 (9%)
881,817.0 (16%)
876,053.0 (10%)
867,595.5 (43%)
866,256.5 (4%)
847,787.5 (23%)
842,235.5 (90%)
830,735.0 (36%)
826,185.5 (10%)
819,099.0 (75%)
815,450.5 (22%)
814,025.5 (11%)
813,973.0 (9%)
796,072.0 (5%)
785,588.0 (27%)
783,484.0 (47%)
781,166.0 (2%)
775,054.0 (28%)
765,337.5 (44%)
758,266.0 (25%)
756,543.0 (4%)
752,060.0 (12%)
747,783.0 (8%)
747,155.5 (5%)
744,310.0 (1%)
743,352.0 (2%)
742,575.0 (3%)
735,833.5 (6%)
716,166.0 (17%)
700,873.5 (11%)
700,565.0 (87%)
694,659.5 (7%)
685,659.5 (3%)
680,046.5 (21%)
673,091.5 (24%)
672,716.5 (5%)
668,973.5 (21%)
662,544.5 (13%)
642,626.0 (22%)
631,966.5 (28%)
631,872.5 (8%)
628,714.5 (15%)
628,655.0 (12%)
626,455.5 (56%)
624,963.5 (23%)
613,351.0 (13%)
608,451.5 (9%)
602,285.5 (11%)
590,174.0 (1%)
588,716.0 (3%)
587,834.0 (49%)
7
3,319.179 (5%)
3,307.314 (15%)
3,306.183 (14%)
3,245.622 (47%)
3,144.419 (71%)
2,996.372 (19%)
2,986.581 (47%)
2,933.300 (46%)
2,929.817 (18%)
2,926.375 (32%)
2,782.254 (15%)
2,744.127 (40%)
2,727.037 (80%)
2,718.594 (57%)
2,705.617 (23%)
2,676.538 (26%)
2,670.668 (6%)
2,640.214 (0%)
2,617.697 (12%)
2,616.430 (38%)
2,488.668 (26%)
2,450.028 (7%)
2,447.570 (21%)
2,429.234 (7%)
2,403.689 (11%)
2,374.611 (80%)
2,364.577 (14%)
2,338.653 (63%)
2,330.973 (12%)
2,307.972 (9%)
2,297.269 (13%)
2,287.329 (33%)
2,286.629 (12%)
2,280.933 (24%)
2,257.503 (18%)
2,256.379 (11%)
2,255.415 (18%)
2,254.565 (15%)
2,254.103 (9%)
2,240.566 (29%)
2,220.634 (10%)
2,141.227 (2%)
2,117.655 (23%)
2,106.105 (5%)
2,083.352 (19%)
2,080.489 (10%)
2,031.177 (11%)
2,028.056 (5%)
2,001.414 (9%)
1,993.920 (6%)
1,988.354 (3%)
1,979.510 (77%)
1,923.468 (27%)
1,914.907 (6%)
1,904.022 (8%)
1,883.679 (1%)
1,874.383 (12%)
1,860.360 (8%)
1,849.200 (62%)
1,840.599 (3%)
1,831.873 (7%)
1,816.037 (42%)
1,809.738 (4%)
1,789.963 (16%)
1,788.107 (18%)
Supporting Table 2. Average quantitation CVs for the proteins identified with 2 to 12 peptides.
Number of peptides
quantified per protein
2
3
4
5
6
7
8
9
10
11
12
Average CVs
0.10
0.11
0.11
0.12
0.11
0.12
0.12
0.09
0.06
0.14
0.10
8
Standard deviation of
average
0.06
0.06
0.09
0.08
0.09
0.06
0.05
0.11
0.04
0.03
0.06
REFERENCES
1.
A. P. Snijders, M. G. de Vos and P. C. Wright, J Proteome Res, 2005, 4, 578-585.
2.
B. Ma, K. Zhang, C. Hendrie, C. Liang, M. Li, A. Doherty-Kirby and G. Lajoie, Rapid Commun
Mass Spectrom, 2003, 17, 2337-2342.
3.
A. Shevchenko, S. Sunyaev, A. Loboda, P. Bork, W. Ens and K. G. Standing, Anal Chem, 2001,
73, 1917-1926.
4.
B. Habermann, J. Oegema, S. Sunyaev and A. Shevchenko, Mol Cell Proteomics, 2004, 3, 238249.
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S. Pevtsov, I. Fedulova, H. Mirzaei, C. Buck and X. Zhang, J Proteome Res, 2006, 5, 3018-3028.
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G. W. Birrell, S. T. Earl, T. P. Wallis, P. P. Masci, J. de Jersey, J. J. Gorman and M. F. Lavin,
Mol Cell Proteomics, 2007, 6, 973-986.
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