Bremmer et al. 1 Dataset 1 - Journal of Biological Chemistry€¦ · 23/11/2011 · Bremmer et al....
37
Dataset 1 The following data provide MS verification of Cdk phosphorylation sites on recombinant protein substrates. Product ion scans of trypsin or Lys-C proteolytic peptides from GST-Acm1, GST-Fin1, and GST-Cdc6 after in vitro phosphorylation with purified Cdk1 are shown. All peptides containing Cdk phosphorylation sites (indicated in red) used in Fig. 1E are shown in unmodified and phosphorylated forms along with standard peptides lacking Cdk consensus sequences used for normalization. Parentheses and “p” indicate phosphorylation of the residue. Only “y” and “b” series ions, and occasionally “a” ions, useful for peptide confirmation and definitive localization of the phosphate group are labeled. As expected from the reported specificity of Cdk1, phosphorylation was limited to the Ser-Pro and Thr-Pro sites within these peptides. The one exception was the Acm1 peptide containing the Cdk site at Ser48, which also showed evidence of low level phosphorylation on Ser37. All spectra were manually interpreted. Product ions used for SRM analyses of Cdc6 peptides with 2 Cdk phosphorylation sites are marked with a green asterisk. Bremmer et al. 1
Transcript of Bremmer et al. 1 Dataset 1 - Journal of Biological Chemistry€¦ · 23/11/2011 · Bremmer et al....
Dataset 1
The following data provide MS verification of Cdk phosphorylation sites on recombinant protein substrates. Product ion
scans of trypsin or Lys-C proteolytic peptides from GST-Acm1, GST-Fin1, and GST-Cdc6 after in vitro phosphorylation
with purified Cdk1 are shown. All peptides containing Cdk phosphorylation sites (indicated in red) used in Fig. 1E are
shown in unmodified and phosphorylated forms along with standard peptides lacking Cdk consensus sequences used for
normalization. Parentheses and “p” indicate phosphorylation of the residue. Only “y” and “b” series ions, and occasionally
“a” ions, useful for peptide confirmation and definitive localization of the phosphate group are labeled. As expected from
the reported specificity of Cdk1, phosphorylation was limited to the Ser-Pro and Thr-Pro sites within these peptides. The
one exception was the Acm1 peptide containing the Cdk site at Ser48, which also showed evidence of low level
phosphorylation on Ser37. All spectra were manually interpreted. Product ions used for SRM analyses of Cdc6 peptides
with 2 Cdk phosphorylation sites are marked with a green asterisk.
Bremmer et al. 1
090722_Acm1 #2312 RT: 33.19 AV: 1 NL: 5.62E3T: ITMS + c NSI d Full ms2 [email protected] [140.00-1700.00]
200 300 400 500 600 700 800 900 1000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
778.
868
722.
313
698.
388
622.
254
550.
727
799.
390
770.
183
622.
904
349.
592
500.
299
552.
457
697.
341
400.
172
494.
364
886.
401
680.
362
442.
319
547.
669
486.
004
613.
441
800.
385
340.
863
987.
481
601.
234
627.
057
912.
513
543.
358
197.
205
391.
445
988.
572
225.
145
869.
376
332.
128
242.
179
921.
420
823.
302
969.
542
K I S L P S F I T P P R N S Kb2
y13 y6
b8b4 b9
y8 y7 y5
b9+
y6+
y7+
y8+
b2+
b8+
y14+2
y13+2
y11+2
y6+2
y7+2
b4+b9
+2
y5+
y14
Acm1 Thr161 Bremmer et al. 2
090722_Acm1 #2392 RT: 34.28 AV: 1 NL: 3.73E4T: ITMS + c NSI d Full ms2 [email protected] [150.00-1780.00]
300 400 500 600 700 800 900 1000 1100m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
556.
578
349.
853
819.
140
550.
878
762.
319
662.
553
698.
310
713.
587
969.
448
770.
661
970.
458
577.
569
613.
298
819.
958
810.
109
340.
692
544.
807
285.
752
442.
245
653.
420
753.
488
952.
531
391.
378
528.
223
841.
316
473.
238
879.
397
663.
378
1067
.358
350.
847
K I S L P S F I (pT) P P R N S Ky6
b4 b9
y10 y7
b4+
y6+y11
+2
y6+2
MH-H3PO4+3
b9-H3PO4+
b9+
y14+2
y14-H3PO4+2
y13-H3PO4+2
y13+2
y10+2
y7-H3PO4+2
y7+
y11y13y14
Acm1 pThr161 Bremmer et al. 3
090722_Acm1 #1440 RT: 20.99 AV: 1 NL: 4.23E4T: ITMS + c NSI d Full ms2 [email protected] [180.00-2000.00]
300 400 500 600 700 800 900 1000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
680.
911
649.
949
644.
119
552.
566
638.
196
488.
515
882.
226
596.
589
756.
385
543.
584
972.
449
634.
766
873.
199
739.
380
682.
637
865.
055
486.
976
818.
219
954.
492
738.
462
378.
941
553.
537
951.
279
973.
482
757.
375
313.
237
809.
218
883.
068
587.
528
937.
441
477.
927
296.
164
489.
422
b8+
y6+
R R S Q I D T D Y A L R R S P I K
y6+2
b2+ b8
+2
y9+2
y15+2
b16+3
b12+2
y6
b2 b8
y9y15
b12 b16
b6+/
b6
Acm1 Ser48 Bremmer et al. 4
090722_Acm1 #1396 RT: 20.36 AV: 1 NL: 4.59E5T: ITMS + c NSI d Full ms2 [email protected] [185.00-2000.00]
400 500 600 700 800 900 1000 1100m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
Rel
ativ
e A
bund
ance
686.
672
676.
619
707.
504
674.
918
644.
001
552.
577
864.
371
638.
091
543.
391
865.
162
488.
507
486.
914
885.
675
1052
.389
378.
851
836.
284
756.
329
596.
524
592.
313
821.
656
972.
452
738.
365
b8+y6
+
R R S Q I D T D Y A L R R (pS) P I K
MH-H3PO4+
b6+2 b8
+2y9
+2
y9-H3PO4+2
y6-H3PO4+2
b6+ a14
+2 b8+H3PO4+
y6
b6 b8
y9
Acm1 pSer48 Bremmer et al. 5
091021_Acm11_T30 #6359 RT: 52.23 AV: 1 NL: 1.16E3T: ITMS + c NSI d Full ms2 [email protected] [265.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1073.433
1587.580
1348.590418.259
1201.608932.257804.354 1461.729 1674.618
989.409657.250
331.317 544.194749.273
1858.795
S D L E V L F Q G P L G S M I S P S K
b7+
y3+
y11+
y12+
y13+
y14+
y7+
y4+
b15+
b16+
b8+
b6+
b5+
b9+
b18+
b6
y11
b18b5
y3y4y13 y12
b7 b8
y7
b15 b16
y14
b9
Acm1 Ser3 Bremmer et al. 6
090722_Acm1 #3508 RT: 49.63 AV: 1 NL: 7.07E2T: ITMS + c NSI d Full ms2 [email protected] [275.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
1153
.440
994.
231
1428
.604
1264
.430
804.
363
1096
.369
932.
301
886.
291
1541
.573
1587
.597
657.
277
498.
181
1656
.622
544.
219
639.
296
1265
.465
786.
458
1343
.704
742.
226
821.
374
400.
346
658.
377
1657
.789
612.
372
331.
253
488.
390
1920
.865
S D L E V L F Q G P L G S M I (pS) P S K
b8+ y10
+
y11+
y13+
y14+b15
+
b16-H3PO4+
b7+
b6+
b5+
y8+
MH-H3PO4+
y6+
y4-H3PO4+
y4+
y3+
b13+
b6
y11 y6
b13b5
y3y4y13 y10
b7 b8
y8
b15 b16
y14
Acm1 pSer3 Bremmer et al. 7
090722_Acm1 #2134 RT: 30.75 AV: 1 NL: 2.90E4T: ITMS + c NSI d Full ms2 [email protected] [145.00-1150.00]
200 300 400 500 600 700 800 900 1000 1100m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
876.
359
991.
317
560.
771
747.
315
496.
281
263.
068
584.
270
555.
152
938.
311
546.
822
992.
403
245.
051
858.
335
392.
122
485.
178
374.
071
654.
212
1007
.332
356.
097
791.
356
748.
326
227.
069
1025
.426
185.
060
729.
331
482.
328
338.
187
438.
805
920.
340
585.
403
878.
318
719.
371
848.
307
293.
059
632.
292
173.
230
L S F H V Y E D Eb6
y2y6
b4 b5
y3y4y5y7
y6+
y4+
b5+
b4+
y5+
y2+
y3+
y7+
y8+
b6+
b7+
b8+
y7+2
MH-H2O+2
b7 b8
y8
Acm1 Standard Peptide 1 Bremmer et al. 8
090722_Acm1 #2620 RT: 37.43 AV: 1 NL: 6.29E4T: ITMS + c NSI d Full ms2 [email protected] [115.00-955.00]
200 300 400 500 600 700 800 900m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
742.
388
629.
280
462.
723
829.
383
201.
104
371.
742
296.
188
482.
254
173.
139
743.
367
335.
174
811.
451
839.
347
461.
147
630.
339
433.
255
234.
220
590.
160
251.
218
I S I F F T S Kb2
y2y6
b3 b4
y3y4y5
y6+
y4+
b2+
b4+
y5+
y2+ y3
+
y7+
y6+2b3-H2O+
y7
Acm1 Standard Peptide 2 Bremmer et al. 9
090722_Acm1 #2052 RT: 29.65 AV: 1 NL: 2.17E6T: ITMS + c NSI d Full ms2 [email protected] [120.00-965.00]
200 300 400 500 600 700 800 900m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
724.
312
467.
708
201.
117
229.
026
637.
299
538.
206
316.
169
397.
090
458.
801
294.
221
706.
302
183.
181
641.
272
468.
377
725.
345
415.
066
423.
257
659.
304
788.
143
D L S V D E F K
y6+
y4+
b2+
b4+
b3+
b6+
y5+
y2+
y3+
b2
y2y6
b3 b4 b6
y3y4y5
Acm1 Standard Peptide 3 Bremmer et al. 10
091021_Fin1_T00 #2629 RT: 30.32 AV: 1 NL: 9.96E3T: ITMS + c NSI d Full ms2 [email protected] [130.00-1570.00]
200 300 400 500 600 700 800 900 1000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
508.
007
501.
942
697.
579
584.
569
641.
105
696.
874
591.
645
706.
541
476.
969
471.
754
893.
489
456.
447
508.
761
894.
464
242.
232
942.
452
427.
762
249.
185
682.
456
520.
631
796.
545
323.
256
231.
230
575.
804
943.
383
277.
114
814.
396
597.
538
358.
038
714.
850
567.
897
390.
155
876.
515
1039
.452
640.
003
715.
724
281.
133
226.
202
186.
294
815.
610
1021
.512
779.
418
E F L K P P M R I S P N K
y3+
y8+2
y8+
y9+
y10+2
y9y11
b2
y10
b3
y8 y3y6
b3+
y11+2
b2+ y6
+
Fin1 Ser54 Bremmer et al. 11
091021_Fin1_T00 #2959 RT: 32.49 AV: 1 NL: 5.89E5T: ITMS + c NSI d Full ms2 [email protected] [140.00-1650.00]
350 400 450 500 550 600 650 700 750m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
507.
961
513.
971
560.
514
582.
543
534.
668
496.
040
737.
340
591.
446
358.
285
592.
211
551.
546
680.
869
688.
150
689.
157
471.
026
493.
934
624.
559
E F L K P P M R I (pS) P N Ky5y7 y6
b12b10
y3
b11
b12-NH3+2
y3+
y9+2
y11+2
b11+2
y10+2
b10-H3PO4+2
MH-H3PO4+3
Fin1 pSer54 Bremmer et al. 12
091021_Fin1_T00 #3689 RT: 37.77 AV: 1 NL: 2.11E4T: ITMS + c NSI d Full ms2 [email protected] [175.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
777.
431
625.
648
630.
318
568.
892
778.
432
890.
494
569.
512
893.
110
631.
347
642.
467
555.
002
698.
894
512.
476
699.
545
1136
.438
389.
300
315.
892
690.
356
1249
.426
503.
661
894.
003
779.
505
1250
.568
1023
.425
1024
.496
495.
125
747.
418
1006
.559
432.
381
832.
667
1396
.460
909.
350
1138
.604
380.
322
1119
.464
1283
.555
242.
290
884.
243
1003
.549
1397
.511
I Q N S D H I T N I I F P T S P T Ky4y7 y6
b12b10 b11
y4+
y8+
y7+
b9+b8
+
y6+
y9+
b10+
b11+
b12+
b10+2
y6+2 y7
+2 b9+2 b12
+2
b11+2
y8y9
b9b8
b2+
b2
Fin1 Ser117 Bremmer et al. 13
091021_Fin1_T00 #3914 RT: 39.50 AV: 1 NL: 1.49E4T: ITMS + c NSI d Full ms2 [email protected] [180.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
857.
363
568.
974
858.
372
625.
371
560.
266
970.
358
625.
987
555.
023
617.
024
642.
333
429.
404
1136
.441
971.
437
933.
566
690.
204
1137
.468
512.
281
690.
889
1249
.519
859.
363
503.
751
839.
313
1023
.516
355.
891
1250
.558
1024
.541
414.
356
759.
309
495.
010
781.
229
1397
.362
1120
.526
242.
321
1083
.444
1283
.724
1396
.418
261.
132
I Q N S D H I T N I I F P T (pS) P T Ky8 y7 y6
b12b10 b11
y4+ y8
+
y9+
b9+
b2+
y7+2
y7+
y6+2
b10+2
b11+2
b9+2/
y7-H3PO4+
b10+
b11+
b12+
b9b2
y4y9
Fin1 pSer117 Bremmer et al. 14
091021_Fin1_T00 #3960 RT: 39.84 AV: 1 NL: 7.27E2T: ITMS + c NSI d Full ms2 [email protected] [180.00-2000.00]
300 400 500 600 700 800 900 1000 1100 1200m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
967.
250
655.
484
876.
329
953.
276
867.
724
682.
315
968.
234
976.
649
909.
787
482.
157
803.
372
802.
450
649.
821
603.
213
858.
686
443.
184
845.
834
539.
108
585.
285
729.
680
230.
117
683.
266
1017
.652
536.
970
744.
654
1131
.521
361.
358
234.
059
1149
.533
442.
310
258.
160
1062
.395
327.
235
409.
376
301.
248
1252
.749
1119
.764
1083
.650
1219
.521
D N V F V R L S M S P L R T T S Q K
y9+b9
+
y15+2
b17+2
b10+
y17+3
y14+2
b2+
a11+2
y14y15y17
a11b10b2 b9
y9
b17
Fin1 Ser36 Bremmer et al. 15
091021_Fin1_T00 #4024 RT: 40.34 AV: 1 NL: 1.47E4T: ITMS + c NSI d Full ms2 [email protected] [185.00-2000.00]
400 500 600 700 800 900 1000 1100 1200m/z
0
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
Rel
ativ
e A
bund
ance
688.
107
681.
954
649.
813
465.
997
916.
275
958.
277
867.
209
1131
.496
708.
655
959.
207
917.
042
868.
307
794.
091
D N V F V R L S M (pS) P L R T T S Q K
MH-H3PO4+3
a11+2
y8+2 y16-H3PO4
+2
y15-H3PO4+2 b17-H3PO4
+2
b10-H3PO4+
y14-H3PO4+2
y8y16 y15
a11b10 b17
y14
Fin1 pSer36 Bremmer et al. 16
091021_Fin13_T30 #2421 RT: 27.51 AV: 1 NL: 2.51E3T: ITMS + c NSI d Full ms2 [email protected] [175.00-2000.00]
200 300 400 500 600 700 800 900 1000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
636.
358
673.
774
958.
208
672.
875
914.
836
642.
468
962.
800
954.
177
963.
478
852.
136
851.
298
816.
948
858.
049
894.
294
817.
689
794.
229
598.
479
859.
006
630.
373
420.
269
675.
696
583.
087
568.
332
785.
727
549.
962
225.
234
763.
773
529.
653
750.
361
733.
474
705.
569
262.
085
290.
219
977.
457
302.
208
473.
157
320.
348
507.
156
421.
379
329.
329
978.
471
402.
173
346.
506
y6+b7
+
b14+2
b2+
H S I Q V T P R R I M S P E C L K
y15+2
y16+2
y14+2
y13+2
b16+3
b15+3
y3+
y4+
y8+
y8y16 y15
b2 b7 b16
y14
b15b14
y13 y6 y4 y3
Fin1 Thr68 / Ser74 Bremmer et al. 17
091021_Fin1_T00 #2664 RT: 30.55 AV: 1 NL: 8.48E4T: ITMS + c NSI d Full ms2 [email protected] [185.00-2000.00]
600 650 700 750 800 850 900 950 1000 1050m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
679.
030
672.
897
694.
093
953.
794
954.
448
695.
122
949.
736
699.
528
667.
160
906.
234
648.
332
945.
204
997.
980
630.
568
735.
638
998.
711
784.
712
849.
231
993.
610
808.
099
889.
214
785.
632
848.
134
y16+2
b16+2
H S I Q V (pT) P R R I M S P E C L Ky16 y15
b6 b16
y14
b14
y12MH-H3PO4+3
y15+2
y16-H3PO4+2
y15-H3PO4+2
y14-H3PO4+2
b14-H3PO4+2
y12+2
y12-H3PO4+2
y11+2
b6-H3PO4+
y11
Fin1 pThr68 / Ser74 Bremmer et al. 18
091021_Fin1_T00 #2649 RT: 30.45 AV: 1 NL: 1.75E6T: ITMS + c NSI d Full ms2 [email protected] [115.00-1385.00]
150 200 250 300 350 400 450 500 550 600 650 700m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
594.
049
396.
418
594.
918
545.
315
187.
162
159.
233
546.
059
397.
063
292.
756
471.
829
D A P F E N D L P R A Ky4y9
b2
b2+
y10+2y10
+3
y5+2 y4
+
y9+2
y10 y5
Fin1 Standard Peptide 1 Bremmer et al. 19
091021_Fin1_T00 #1509 RT: 22.72 AV: 1 NL: 5.29E5T: ITMS + c NSI d Full ms2 [email protected] [110.00-1365.00]
200 250 300 350 400 450 500 550 600m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
438.
475
495.
796
395.
926
496.
416
432.
714
323.
185
297.
128
341.
141
551.
481
523.
192
559.
966
428.
243
373.
715
541.
087
519.
395
452.
536
338.
258
406.
700
423.
934
367.
717
524.
240
213.
074
487.
041
E T Q S L D R P Q F Kb3
b5+
y6+2
y4+
y6 y8+2
y7+2
b3-H2O+
b4-H2O+
y4y7y8
b4 b5
Fin1 Standard Peptide 2 Bremmer et al. 20
091021_Fin1_T00 #1511 RT: 22.72 AV: 1 NL: 6.03E3T: ITMS + c NSI d Full ms2 [email protected] [180.00-1395.00]
200 300 400 500 600 700 800 900 1000 1100 1200m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
851.
282
673.
613
581.
997
852.
404
1079
.405
964.
404
674.
324
590.
975
682.
438
531.
294
1180
.431
513.
181
664.
954
1080
.511
490.
331
604.
393
1181
.521
1105
.525
573.
069
1218
.372
660.
644
260.
348
1235
.320
418.
255
303.
207
400.
173
1162
.483
834.
349
312.
071
853.
447
833.
435
532.
200
389.
266
875.
358
202.
172
976.
236
761.
380
778.
276
468.
202
993.
338
892.
261
1062
.479
742.
285
947.
410
725.
201
1061
.440
S N T D L C S N T E L K
b4+
y4+
y3+
y2+
b2+ b3
+y5
+
y7+
y8+ y9
+
y10+b5
+
b7+ b8
+ b9+
b11+
y6-H2O+
y10-H2O+2
b2
y2y6
b3 b4 b5
y3y4y5
b7 b8 b9 b11
y7y8y9y10
Fin1 Standard Peptide 3 Bremmer et al. 21
091021_Fin1_T00 #3559 RT: 36.76 AV: 1 NL: 2.66E4T: ITMS + c NSI d Full ms2 [email protected] [100.00-840.00]
200 250 300 350 400 450 500 550 600 650 700m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
601.
392
488.
352
200.
191
228.
153
602.
438
341.
266
406.
201
489.
434
724.
418
389.
271
513.
097
260.
201
183.
176
440.
209
482.
208
229.
224
569.
385
201.
240
470.
047
342.
306
357.
867
725.
610
714.
396
552.
280
324.
384
N L L V E L Kb2
b4+ y6
+
y2y6y5
+
y4+
y3+y2
+
b2+
b3+
b5+
b3 b4 b5
y3y4y5
Fin1 Standard Peptide 4 Bremmer et al. 22
101003_63-T05 #11689 RT: 40.80 AV: 1 NL: 1.69E5T: ITMS + c NSI d Full ms2 [email protected] [140.00-1665.00]
200 250 300 350 400 450 500 550 600 650 700 750 800m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
713.
216
714.
051
639.
631
774.
737
200.
150
539.
693
228.
181
489.
038
541.
908
582.
097
375.
336
524.
554
513.
629
640.
463
704.
144
685.
850
475.
812
354.
468
775.
548
629.
516
183.
116
b2+a2
+b3
+ y9+2
y6+2
y12+2
y13+2
y11+2
y10+2
N L F D D A P A T P P R P L Ky9y10y11y12y13 y6
b2 b3
Cdc6 Thr23 Bremmer et al. 23
101003_63-T05 #12740 RT: 43.96 AV: 1 NL: 4.85E5T: ITMS + c NSI d Full ms2 [email protected] [145.00-1745.00]
200 250 300 350 400 450 500 550 600 650 700 750 800 850m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
753.
228
754.
072
679.
604
528.
994
561.
344
622.
063
200.
147
228.
137
564.
541
814.
738
680.
595
354.
479
375.
296
655.
933
744.
142
652.
372
707.
499
815.
693
502.
477
N L F D D A P A (pT) P P R P L K
y12+2
y13+2
y11+2
b2+a2
+
b3+y6
+2
y9+2
y10+2
y9y10y11y12y13 y6
b2 b3
Cdc6 pThr23 Bremmer et al. 24
101003_63-T05 #14022 RT: 47.62 AV: 1 NL: 5.66E5T: ITMS + c NSI d Full ms2 [email protected] [175.00-1360.00]
300 400 500 600 700 800 900 1000 1100m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
870.
649
1070
.735
871.
611
664.
592
458.
304
1071
.727
655.
710
276.
276
476.
323
872.
547
1072
.735
983.
736
345.
263
373.
409
573.
435
272.
281
973.
628
430.
364
886.
626
259.
252
984.
794
1052
.688
277.
264
773.
566
868.
660
527.
159
955.
666
477.
307
774.
594
374.
490
755.
582
574.
461
329.
324
686.
458
1038
.775
647.
707
605.
478
540.
332
F Q S L P L S L S T P Ry3y5y8 y7y9 y2
b2 b4
b2+
y3+
b4+
b6+
b7+/y7
+b8
+
b9+
y10+
y9+
y8+
y2+
b5+/y5
+
b5 b6 b7 b8 b9
y10
Cdc6 Thr135 Bremmer et al. 25
101003_63-T05 #14972 RT: 50.29 AV: 1 NL: 4.67E4T: ITMS + c NSI d Full ms2 [email protected] [185.00-1440.00]
300 400 500 600 700 800 900 1000 1100m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
950.
561
951.
552
1150
.635
458.
307
1151
.613
276.
195
952.
586
476.
293
695.
683
655.
608
345.
278
704.
575
540.
218
272.
302
740.
417
1063
.589
1152
.612
653.
368
430.
287
1056
.671
363.
277
1064
.604
853.
582
886.
667
973.
564
741.
385
1038
.637
1132
.675
932.
645
541.
242
887.
589
773.
544
477.
303
277.
237
852.
640
974.
591
567.
469
640.
261
328.
273
809.
790
F Q S L P L S L S (pT) P Ry3y5y8 y7y9 y2
b2 b4 b7 b8 b9
y10+
y9+
y8+
y7+y6
+y5
+y4+y2
+ y3+
y4y6y10
b2+
b3+
b4+
b3
b7+ b8
+
b9+
Cdc6 pThr135 Bremmer et al. 26
101003_63-T05 #10858 RT: 38.43 AV: 1 NL: 3.06E4T: ITMS + c NSI d Full ms2 [email protected] [205.00-1590.00]
300 400 500 600 700 800 900 1000 1100 1200 1300m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
874.
568
773.
530
1336
.682
1337
.682
704.
424
771.
770
1189
.650
774.
502
1088
.611
973.
574
1190
.616
669.
048
805.
545
974.
611
1089
.605
705.
475
605.
375
1205
.589
242.
221
389.
340
373.
443
770.
527
1318
.694
876.
586
659.
629
460.
384
806.
492
547.
301
490.
357
893.
599
1031
.667
975.
597
1118
.683
657.
496
1188
.543
1032
.665
1339
.452
390.
404
956.
759
344.
415
539.
303
L Q F T D V T P E S S P E K
y10+
y9+
y8+
y7+
y5+
y11+
y12+
y4+y3
+b2+
b3+
b4+ b5
+
b6+
b7+
b9+ b10
+
b11+
b2 b4 b6 b7b5 b9b3 b10 b11
y4y8y9 y3y5y7y10y11y12
Cdc6 Thr39 / Ser43
*
Bremmer et al. 27
100824_2T0 #11218 RT: 38.74 AV: 1 NL: 9.77E3T: ITMS + c NSI d Full ms2 [email protected] [215.00-1670.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
954.
427
853.
410
771.
666
1416
.602
805.
476
704.
414
854.
394
1417
.614
1053
.471
1168
.468
1269
.476
709.
116
1270
.508
686.
396
1054
.492
762.
654
605.
293
956.
411
936.
411
389.
331
627.
266
855.
426
242.
146
874.
458
1398
.593
756.
326
587.
349
1031
.536
676.
420
1170
.487
540.
222
373.
361
490.
242
463.
290
1251
.527
1118
.698
1300
.673
1188
.679
1013
.578
445.
309
1318
.594
1419
.523
364.
195
L Q F T D V T P E S (pS) P E Kb2 b4 b6 b7b5 b9b3 b10
y4y8y9 y3y5y7y10y11y12
y5+
y4+y3
+b2+ b3
+
b4+
b5+
y7+
y8+
y9+ y10
+y11
+
y12+
b7+
b6+
b9+
b10+
Cdc6 Thr39 / pSer43
*
Bremmer et al. 28
100823_1T30 #11168 RT: 37.29 AV: 1 NL: 2.13E4T: ITMS + c NSI d Full ms2 [email protected] [215.00-1670.00]
400 600 800 1000 1200 1400 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
693.
440
580.
312
1078
.574
863.
572
811.
720
1079
.587
965.
492
1252
.609
934.
627
1165
.621
694.
417
581.
289
466.
273
1416
.607
1253
.645
777.
502
1166
.612
1060
.626
773.
513
1417
.635
1080
.596
1483
.743
929.
439
704.
425
406.
207
1269
.564
303.
325
467.
293
562.
238
1355
.616
544.
268
865.
565
1270
.590
688.
374
373.
296
619.
428
967.
541
1148
.682
1485
.818
242.
206
1511
.942
L Q F T D V (pT) P E S S P E K
y3+
b2+
y7+
y11+
y12+
y9+
y8+
b2
y8y9 y3y7y11y12
b6+
b6
y4+
y4
Cdc6 pThr39 / Ser43
*
Bremmer et al. 29
100824_2T0 #10780 RT: 37.49 AV: 1 NL: 2.67E4T: ITMS + c NSI d Full ms2 [email protected] [235.00-1810.00]
500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
Rel
ativ
e A
bund
ance
1040
.668
757.
461
740.
410
739.
439
1042
.625
1139
.710
1140
.684
758.
442
1267
.715
722.
398
1068
.594
881.
358
1268
.689
830.
567
721.
416
641.
336
967.
588
1338
.815
1069
.674
640.
365
658.
393
882.
034
1141
.700
623.
357
512.
323
1339
.766
495.
296
831.
507
1426
.827
759.
472
530.
245
826.
938
1022
.581
1250
.753
968.
631
G S L N S A Q V P L T P T T S P V Kb6
y10
b7+ y8
+
b8+
b6+
y10+
y11+
y12+
y13+b10
+
b11+
y7+
y12 y11y13 y8 y7
b7 b8 b10 b11
Cdc6 Thr368 / Ser372
*
*
Bremmer et al. 30
100824_2T0 #11219 RT: 38.74 AV: 1 NL: 2.70E4T: ITMS + c NSI d Full ms2 [email protected] [245.00-1890.00]
500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
Rel
ativ
e A
bund
ance
1120
.601
757.
437
740.
438
1122
.554
739.
462
809.
455
1219
.627
1068
.601
758.
458
910.
452
722.
383
890.
271
1220
.662
810.
499
921.
692
1331
.640
1123
.548
967.
689
1330
.636
1050
.580
641.
325
1023
.596
721.
432
1022
.606
658.
364
759.
518
640.
315
1221
.708
1418
.827
811.
539
623.
395
512.
344
1419
.807
1332
.699
1506
.885
495.
265
530.
070
922.
626
1505
.690
704.
406
1201
.683
b7+
y8+
b6+
y10+
y11+
y7+
y13+b10
+
b11+
y9+
b8+
G S L N S A Q V P L T P T T (pS) P V Kb6
y10 y9y11y13 y8 y7
b7 b8 b10 b11
Cdc6 Thr368 / pSer372
*
Bremmer et al. 31
101003_62T30 #11716 RT: 40.38 AV: 1 NL: 2.95E4T: ITMS + c NSI d Full ms2 [email protected] [245.00-1890.00]
400 600 800 1000 1200 1400m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
1120
.636
757.
449
740.
462
1122
.632
739.
491
890.
320
1219
.661
758.
512
722.
420
921.
791
1220
.646
1330
.766
1050
.606
1123
.602
1022
.761
967.
728
823.
818
640.
433
658.
422
876.
252
430.
313
512.
335
495.
306
705.
421
1430
.815
1447
.824
1534
.892
1329
.794
1419
.849
814.
777
1505
.918
513.
271
y10+
y11+y7
+
y13+b10
+
b11-H3PO4+
y9+
b8+
G S L N S A Q V P L (pT) P T T S P V K
b7+
y10 y9y11y13 y8 y7
b7 b8 b10 b11
Cdc6 pThr368 / Ser372
*
Bremmer et al. 32
100824_2T0 #11156 RT: 38.57 AV: 1 NL: 8.56E3T: ITMS + c NSI d Full ms2 [email protected] [255.00-1970.00]
500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
1200
.560
1201
.539
757.
423
740.
395
739.
479
921.
233
1299
.601
809.
432
722.
419
960.
716
1102
.598
758.
431
961.
504
1182
.522
1300
.645
1050
.604
1410
.650
721.
414
913.
534
1103
.712
1411
.716
912.
274
810.
464
704.
380
641.
405
510.
265
850.
515
640.
293
1498
.623
512.
347
1281
.681
623.
412
495.
269
1499
.789
G S L N S A Q V P L (pT) P T T (pS) P V Ky10
+
y11+
y7+
y13+
y10-H3PO4+
b8+
b7+
b11-H3PO4+
y4+ y15
+2
y10y11y13 y4y7
b7 b8 b11
y15
Cdc6 pThr368 / pSer372
*Bremmer et al. 33
101003_63-T05 #15125 RT: 50.73 AV: 1 NL: 6.61E5T: ITMS + c NSI d Full ms2 [email protected] [165.00-1310.00]
300 400 500 600 700 800 900 1000 1100m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50R
elat
ive
Abu
ndan
ce
1054
.672
907.
626
435.
349
242.
202
631.
446
1056
.635
639.
641
527.
943
908.
615
861.
551
389.
329
548.
431
850.
605
1121
.684
592.
100
1036
.599
862.
507
763.
527
436.
452
843.
582
519.
131
243.
203
1122
.736
288.
387
1008
.664
748.
556
583.
128
909.
632
626.
398
764.
516
990.
652
661.
414
446.
401
1104
.712
429.
372
730.
424
390.
342
L Q F G S Q S I F L R
y7+
b7+y2
+
b3+
y9+
b10+y4
+
y3+
y6+
y8+
b6+b4
+
b8+
b9+
y9 y4y7
b3 b4
y8 y6 y3 y2
b7 b8 b9 b10b6
Cdc6 Standard Peptide 1
*
*
Bremmer et al. 34
101003_63-T05 #10535 RT: 37.52 AV: 1 NL: 1.90E5T: ITMS + c NSI d Full ms2 [email protected] [180.00-1405.00]
300 400 500 600 700 800 900 1000 1100 1200m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
613.
441
556.
388
714.
518
837.
489
679.
325
827.
589
780.
501
715.
554
614.
410
819.
498
557.
371
990.
642
566.
322
762.
509
838.
591
781.
519
661.
371
991.
647
548.
284
1189
.569
538.
370
1190
.639
385.
384
992.
687
716.
511
459.
376
403.
272
615.
451
1103
.786
290.
201
272.
258
1246
.700
357.
382
477.
436
972.
781
934.
550
1031
.600
346.
286
1247
.541
857.
281
1089
.712
914.
826
404.
395
1104
.653
1032
.606
329.
453
1172
.829
S D S L Y I T G P P G T G Ky7
+y6
+
b6+
b7+
y10+
y9+
y8+
b13+
b8+
y11+
b12+
b10+b9
+y5
+b4
+b3
+
b5+
y9 y5y7
b3 b4
y8 y6y10
b7 b8 b9 b10b6b2 b5 b12 b13
y11
Cdc6 Standard Peptide 2
*
*
Bremmer et al. 35
101003_63-T05 #11696 RT: 40.83 AV: 1 NL: 1.18E6T: ITMS + c NSI d Full ms2 [email protected] [225.00-1745.00]
400 600 800 1000 1200 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
elat
ive
Abu
ndan
ce
1387
.717
343.
327
1388
.717
1214
.652
1370
.713
987.
610
456.
392
1215
.651
988.
620
1274
.640
743.
503
1369
.747
1100
.688
1275
.597
584.
384
847.
866
516.
329
726.
490
873.
531
457.
450
1389
.706
699.
578
840.
524
786.
577
1146
.548
989.
698
417.
363
585.
422
1556
.855
944.
617
527.
300
1031
.700
613.
380
286.
285
399.
340
630.
455
927.
669
1427
.850
1557
.806
1539
.000
531.
381
1428
.857
268.
424
S S E L V N L N S S D G A L P A R
y7+
y3+
b6+
b7+
y10+
y9+
y8+
b10+y5
+b4+
b9+
b5+
y4+
y6+
y11+
y12+
b11+
b13+
b14+y9 y5y7
b4
y8 y6y10
b7 b9 b10b6b5 b11 b13
y11 y4 y3y12
b14
Cdc6 Standard Peptide 3
*
*
Bremmer et al. 36
101003_63-T05 #12273 RT: 42.55 AV: 1 NL: 3.90E5T: ITMS + c NSI d Full ms2 [email protected] [150.00-1195.00]
300 400 500 600 700 800 900 1000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
681.
446
518.
582
500.
305
923.
578
794.
570
682.
467
924.
557
519.
281
582.
006
1034
.613
795.
577
663.
425
387.
336
482.
283
389.
392
240.
231
905.
582
1035
.589
792.
507
369.
321
645.
393
1016
.539
276.
255
519.
990
925.
626
683.
414
887.
576
472.
388
796.
596
572.
574
359.
345
454.
343
713.
574
390.
412
582.
963
635.
554
774.
490
736.
836
571.
314
G S I E I Y E L E K
y7+
y3+
b6+
b7+y2
+
y5+
b4+
b5+y4
+
y6+
b9+
b8+
y5y7
b4
y6
b7 b9b6b5
y4 y3
b8
y2
Cdc6 Standard Peptide 4 Bremmer et al. 37
