Supporting Information for:
Ion mobility-mass correlation trend line separation of glycoprotein digests
without deglycosylation
Hongli Li1, Brad Bendiak2, William F. Siems1, David R. Gang3, and Herbert H. Hill, Jr.1, *
1
Department of Chemistry, Washington State University, Pullman, Washington, US
2
Department of Cell and Developmental Biology, Program in Structural Biology and Biophysics, University of
Colorado, Health Sciences Center, Anschutz Medical Campus, Aurora, Colorado, US
3
Institute of Biological Chemistry, Washington State University, Pullman, Washington, US
Table S1. Additional instrumental parameters of Synapt G2 HDMS
Parameters
ESI Voltage (KV)
Desolvation Gas
Sample Cone Voltage (V)
Extraction Cone Voltage (V)
Source Temperature (°C)
Helium Gas (IMS mode)
Argon Gas (Trap &Transfer cell)
Positive mode
3.2
Nitrogen, 200°C, 600 L/hr
40.0
4.0
150
180 mL/min
2 mL/min
Negative mode
2.25
Nitrogen, 200°C, 600 L/hr
40.0
4.0
150
180 mL/min
2 mL/min
S-1
Table S2. Identified singly and doubly charged peptides
corresponding to human α-1-acid glycoprotein
m/z (+1)
Peptide Sequence
m/z (+2)
Peptide Sequence
262.1
DK
339.7
QEEGES
304.2
ER
348.7
EYQTR
357.3
IPK
481.2
DKCEPLEK
363.3
DTK
497.8
TEDTIFLR
418.3
ITGK
572.8
SDVMYTDWK
696.3
EYQTR
709.8
TLMFGSYLDDEK
994.5
TEDTIFLR
723.3
TYMLAFDVNDEK
961.5
DKCEPLEK
843.4
EQLGEFYEALDCLR
1112.5
SDVVYTDWK
1012.5
EQLGEFYEALDCLRIPK
1144.5
SDVMYTDWK
1418.7
TLMFGSYLDDEK
1445.7
TYMLAFDVNDEK
1582.8
CEPLEKQHEKER
Table S3. Identified singly and doubly charged peptides
corresponding to human antithrombin III
m/z (+1)
Peptide Sequence
m/z (+1)
Peptide Sequence
m/z (+2)
Peptide Sequence
175.1
R
761.4
VWELSK
400.7
IPEATNR
322.2
FR
800.4
IPEATNR
456.3
LPGIVAEGR
338.2
YR
839.4
FDTISEK
459.3
LPGIVAEGR
418.3
SLAK
850.4
FSPENTR
588.3
SLNPNRVTFK
460.3
KANK
860.5
LQPLDFK
655.3
DDLYVSDAFHK
462.2
TEGR
911.5
LPGIVAEGR
670.8
TSDQIHFFFAK
503.3
GLWK
917.5
RVWELSK
715.9
VAEGTQVLELPFK
579.3
LFGDK
961.5
SSKLVSANR
925.0
EQLQDMGLVDLFSPEK
659.4
LVSANR
S-2
Fig. S1 MS/MS spectra of +2 charged glycopeptides from trend line III in Fig. 1 in the paper.
The collision energy of 50V, 42V and 33V were used for the precursor ions from top to the
bottom, respectively. Note: underlined peaks are precursor ions.
0.25mg/ml in 0.1%FA ESI,quadrupole res at 4.9, trap ce50V
1+
366.1
a
100
%
1+
186.1
2+
2+
367.1 1+
2+
1516.7 1699.8
1+
1+
528.2 1+
1+
2+
1+
1253.1
1+
731.1 893.31055.4
1435.7 1611.9
1815.0 1983.1
250
500
750
1000
0.25mg/ml in 0.1%FA ESI,quadrupole res at4.9, trap ce42V
1+
b
1250
0
286.2
168.1 1+ 367.1 500
204.1
508.3
368.1100
800
700
600
1+
1+
972.4
839.4
1+
893.3
887.4
c
366.1
1+
288.2
1197.0
1197.5
1175.6
915.3 980.51+
2+
1200.0
1055.4
1116.0
981.5
900
629.3
508.3
0
1022.5
%
%
168.1
0
838.4
726.4
762.4
1100
1000
1200
2+
1298.5
1302.0
1300
m/z
2+
1379.5 1562.6
1400
1500
1600
1750
1022.5
1140.5
1800
2000
m/z
m/z
886.4
887.4
200 200 400
600
500
725.4
600
400
800
600
700
800
900
1000
1+
1+1100
m/z
1300
2+
1379.6
1022.5
885.4
551.3
1380.1
2+
1298.51379.1
886.4
1+
1380.6
2+
1+ 972.5
367.1
1383.6
1+
1+
2+
1116.0
1+
887.4
1+
731.31022.5
875.5 893.3
1197.0 1378.6 1384.1
528.2
368.1
299.2
304.2
1+
286.2
696.3
1+
2+
725.4
591.3
2+
979.5
1+
200250 400 500 600 551.3
1000 1250
1200
885.4
750 800 1000
%
100
m/z
2000
838.4
0.25mg/ml in 0.1%FA ESI,quadrupole res at 4.9,549.3
trap ce33V
100
1750
1+
1+
100 528.2
1+
508.3
731.3
725.4
304.2
1+
885.4 732.3
1+
604.2
551.3
512.2 529.2
1+
1+
1+
747.3
690.2
569.2 657.3 886.4
%
0
1500
0.25mg/ml in 0.1%FA ESI,quadrupole res at4.9, trap ce42V
366.1
288.2
%
100
100
1334.1
1070.5
690.2
138.1 204.1
0
2+
2+
1+
1+
168.1
1+
1+
1055.4
1000800 1200 1000
1400
1200
1600
1200
1800
1400
m/z
m/z
288.2
100
508.3
%
725.4
Spectrum rough analysis for a: 304.2
549.3
Neutral loss of Hexose (Hex) includes 366.1-204.1;
528.2-366.1;
690.2-528.2; 893.3-731.1; 1055.4-893.3; 1334.1885.4
551.3
1253.1 and 1516.7-1435.7.
886.4
591.3 838.4
Neutral loss of N-acetylhexosamine
(HexNAc) includes 731.1-528.2; 893.3-690.2 and 1815.0-1611.9.
286.2
120.1
738.1
887.4 1022.5
1140.5
Spectrum rough analysis for b:
0
m/z
Neutral loss of Hexose (Hex)
690.2-528.2;
893.3-731.1;
1055.4-893.3 and
200 includes
400366.1-204.1;
600 528.2-366.1;
800
1000
1200
1400
1197.0-1116.0.
Neutral loss of N-acetylhexosamine (HexNAc) includes 731.1-528.2; 893.3-690.2; 1175.6-972.4 and 1298.5-1197.0.
Spectrum rough analysis for c (m/z 1379.6 is precursor ion):
Neutral loss of Hexose (Hex) includes 366.1-204.1; 528.2-366.1; 690.2-528.2; 893.3-731.1; 1055.4-893.3 and
1197.0-1116.0.
Neutral loss of N-acetylhexosamine (HexNAc) includes 731.1-528.2; 893.3-690.2; 1175.6-972.4 and 1298.5-1197.0.
S-3
Fig. S2 MS/MS spectra of +3 charged glycopeptides from trend line IV in Fig. 1 in the paper.
The collision energy of 38V and 40V were used for the ions from top to the bottom, respectively.
Note: underlined peaks are precursor ions.
0.25mg/ml in 0.1%FA ESI,quadrupole res at 5.0, trap ce35V
100 a
138.1
1+
1+
366.1
3+
942.5
1+
604.3
1+
943.1
%
186.1
942.1
1+
204.1
1+
168.1
528.2
1+
320.1
0
367.1
200
1+
733.4
1+
549.3
400
605.3
1+
707.4
600
2+
1401.5
2+
2+
996.8 2+ 1215.6
1400.5 1413.2
2+
936.8
1137.9 2+
1414.7
1165.0 1272.1
800
0.25mg/mlinin0.1%FA
0.1%FAESI,quadrupole
ESI,quadrupole
at4.9,
ce42V
0.25mg/ml
resres
at 4.9,
traptrap
ce40V
3+
1055.5
3+
1056.7
1000
2+
1365.2
1200
m/z
1400
1+
288.2
366.1
100
100
366.1
100 b
508.3
%
% %
304.2
168.1
286.2367.1
1+
299.2
1+
367.1
0 168.1
250
500
200
400
204.1
288.2
1+ 738.1
1+
731.3
1044.5 1055.4
528.2
0
0
100
1+
725.4
549.3
2+
1545.3
885.4
551.3
591.3 838.4 886.4
3+
750
368.1
1000
600
1250
800
2+
2174.0
2+
1625.7
887.4
1627.1
1624.7
1022.5
2072.4
2+
1990.9
1140.5
1500
1000
1750
1200
2000
1379.5 1562.6
2250
1400
200
400
600
800
1000
1200
1400
1600
Spectrum rough analysis for a:508.3 725.4
304.2
Neutral loss of Hexose (Hex)
528.2-366.1 and 996.8-942.5.
288.2 includes 366.1-204.1;
885.4
100
551.3
%
2+
2255.0
1800
m/z
m/z
m/z
886.4
%
Spectrum rough analysis
286.2 for b: 508.3
725.4
Neutral loss of Hexose (Hex)
includes 366.1-204.1;
304.2
887.4 528.2-366.1; 2072.4-1990.9 and 2255.0-2174.0.
1140.5
Neutral loss of N-acetylhexosamine (HexNAc) includes
731.1-528.2 and 2174.0-2072.4.
885.4
551.3
0
m/z
250
500
750
1000
1250
886.4
1500
1750
2000
2250
1400
1600
1800
286.2
887.4 1022.5
0
200
400
600
800
1000
1200
m/z
S-4
Fig. S3 (a) Extracted mass spectrum of trend line IV in Fig. 5 for human antithrombin II. (b)
Mass spectrum with expanded region from m/z 550 to m/z 750.
pos sen,0.45mg/ml in 0.1%FA 50/50ESI,40V650m/s,450us delay on,mass50-2000
100
605.7
634.7
604.7
a
488.8
664.7
517.8 575.7
709.6
752.6 810.6
839.5
928.5 956.5
1071.4
477.8
%
1112.4 1173.4
0
400
500
600
700
pos sen,0.45mg/ml in 0.1%FA 50/50ESI,40V650m/s,450us delay on,mass50-2000
100
800
900
1000
m/z
1100
605.7
604.7
b
575.7
574.7
634.7
663.6
633.7
664.7
635.7 662.6
680.6
606.7
603.7
650.7
679.6
621.7
592.7
692.6
709.6 722.6
739.6
723.6
737.6
563.7
576.7
%
562.7
620.7
577.7
0
560
580
600
620
640
660
680
700
720
740
m/z
S-5
Fig. S4 Extracted mass spectra corresponding to different trend lines in Fig.8 for human α-1acid glycoprotein in the paper. The inset on the right in each spectrum illustrates the isotopic
patterns of -2, -3 and -4 charged ions.
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min
%
100
0
451.3 Trend line I: -1 Charged Peptides
723.3
506.3 580.3
311.1
883.4
596.3
836.4
332.2
885.4 1020.5
400
361.2
100
%
600
0
509.7
583.8
589.8 698.8
1104.5
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min
400
600
800
1000
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min
100
1208.5
Trend line III: -2 Charged Glycopeptides 1208.0
1209.0
%
1209.5
1262.5
0
1391.0
0
1426.5
1609.1
m/z
1200
1208.5
1209.0
%
100
m/z
1200
1000
Trend line II: -2 Charged Peptides
436.2
352.2
800
1209.5
1208
1746.8
1209
1883.4
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min
1200
1300
1400
1500
1600
1700
1800
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min
%%
0
0
1429.6
1163.5
1430.3
1428.9
1164.2
1164.8 1348.6
1552.0
%
1208.5 1551.3
100 Trend line IV: -3 Charged Glycopeptides
100
1551.3
1551.0
1208.0 1209.0
100
1209.5
1900
m/z
m/z
1551.6
1552.0
1552.3
1257.4
1552.6
0 1210.0
m/z
1624.1
1262.5
1551
1552
1207.6 1788.8
m/z
1050 14001100
1150
1200 1800
1250 1900 m/z
1300
1500
1600
1700
1000
1200
Trend line V: -4 Charged Glycopeptides 100
1163.2 1163.5
1163.5
1163.0
1163.7
100
1164.0
1163.0 1163.7
1071.9
1164.2
1072.4
1164.0
0
m/z
1162.7
1071.4 1072.7
1163
1164
1164.5 1199.7
1254.5
1010.9
0
m/z
1000
1050
1100
1150
1200
1250
%
%
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min
S-6
Fig. S5 MS/MS spectra of -2 charged glycopeptides from trend line III in Fig. 8 for human α-1acid-glycoprotein in the paper. The collision energy of 68V, 57V and 50V were used for the
precursor ions from top to the bottom (Fig. a-c), respectively. Note: underlined peaks are
precursor ions.
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min , trap ce 68V
624.3
a
100
1861.4
1883.4
1860.9 1883.9
%
1843.7
580.3
1884.4
625.3
1640.6
179.1
694.3
0
500
887.5
1000
1885.4
1500
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min , trap ce 57V
100
1884.9
1130.7 1478.5
2000
m/z
877.4
b
1391.0
1391.5
424.1
%
878.4
179.1
290.1
0
250
551.2
500
731.3
750
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min , trap ce 50V
100
884.4
1000
1390.5 1392.1
1382.5
1392.6
1382.0
1309.5
1250
1750
m/z
877.4
c
1208.5
424.2
878.4
%
179.1
0
1500
1843.7
1641.6 1845.6
250
551.2
500
860.4
1208.0
1199.9
1135.4
951.3
750
1000
1209.0
1478.5
1276.4 1480.6
1250
1481.5
1500
1750
m/z
Spectrum rough analysis:
a. Neutral loss of Hexose (Hex) includes 1640.6-1478.5 and 1843.7-1663.6.
Neutral loss of N-acetylhexosamine (HexNAc) includes 1681.6-1478.5 and 1843.7-1640.6.
b. Neutral loss of Fucose (Fuc) includes 877.4-731.3; 1391.0-1318.0 and 1382.5-1390.5.
Neutral loss of Hexose (Hex) includes 1640.6-1478.5; 1663.6-1501.5; 1783.6-1621.6 and 1843.7-1681.6.
Neutral loss of N-Acetylhexosamine (HexNAc) includes 1478.5-1275.4 and 1843.7-1640.6.
c. Neutral loss of Fucose (Fuc) includes 877.4-731.3; 1199.5-1126.4; 1208.5-1135.4 and 1864.7-1718.6.
Neutral loss of Hexose (Hex) includes 731.3-551.2; 1095.4-933.3; 1113.4-951.3; 1418.5-1256.5 and 1718.61556.5. Neutral loss of N-acetylhexosamine (HexNAc) includes 1113.4-951.5 and 1478.5-1275.4.
S-7
Fig. S6 MS/MS spectra of -3 charged glycopeptide from trend line IV in Fig. 8 for human α-1acid-glycoprotein in the paper. The collision energy of 50V was used. Note: underlined peaks are
precursor ions.
neg sen,0.5mg/ml in 50/50 methanol and water, 3ul/min , trap ce 50V
1551.6
100
959.5
1551.3
957.4
%
960.5
925.4
913.5
907.5
128.0 424.1 665.3
0
500
991.4
1545.3
1544.9
992.4
1000
1847.8
1552.3
1848.2
1545.6 1552.6
1539.7
1846.7 1849.2
1553.7 1849.7
1830.7 1850.2 2208.8
2211.8
2005.8
1500
2000
m/z
Spectrum rough analysis:
Neutral loss of Fucose (Fuc) includes 1847.8-1774.7, 1831.2-1858.2 and 959.5-795.4
Neutral loss of Hexose (Hex) includes 844.4-682.3
Neutral loss of N-acetylhexosamine (HexNAc) includes 2208.8-2005.8
neg sen ,0.45mg/ml in 50/50esi,40V,650m/s,450us delay on
Fig. S7 Mass spectrum of the noise region labeled in Fig. 9 for human antithrombin III.
200.3
239.8
%
100
0
m/z
200 250 300 350 400 450 500 550 600 650 700
649.6
649.3650.0
%
100
541.6 584.9
636.3
650.3
0
m/z
200 250 300 350 400 450 500 550 600 650 700
S-8
Fig. S8 Extracted mass spectra corresponding to different trend lines in Fig. 9 for human
antithrombin III in the paper.
pos sen,1mg/ml in in 50/50 methanol and water, 3ul/min , 40v,650m/s, 450us delay
387.2
Trend line I: -1 Charged Peptides
623.3 686.4
416.3
376.2
506.3
584.3
995.5
747.3
255.2
840.5 992.5 1014.4
neg sen ,0.45mg/ml in 50/50esi,40V,650m/s,450us delay on
0
m/z
200
400
600
800
1000
%
100
824.5
828.9
Trend line II: -2 Charged Peptides
854.0
824.1
898.0
376.8
300.8803.3
495.7
100 260.9
%%
100
544.8 634.7
0
0600
200
800
400
1000
600
882.4
1200
800
1400
1000
m/z
m/z
760.4 Trend line III: -3 Charged Glycopeptides
1396.9
760.1 760.7
100
1396.5
1397.2
650.0
1397.5
1396.2
761.1
750.7
848.0
1396.9
887.0 1036.4 0
m/z
1397
1436.9
0
m/z
pos sen,1mg/ml in in 50/50 methanol and water, 3ul/min , 40v,650m/s, 450us delay
600
800
1000
1200
1400
100
%
%
neg sen ,0.45mg/ml in 50/50esi,40V,650m/s,450us delay on
Trend line IV: Unidentified compounds
%
100
0
100
664.6 735.6 764.6
with systematic mass difference
662.6
835.6 864.5
935.5
620.6
1005.5
600
552.7
%
554.7
0
600
700
800
634.7
669.3
713.9
700
900
1000
1100
m/z
882.4
882.9
838.6
800
883.4 978.5
900
1000
1036.5 1126.5
1100
m/z
S-9
Fig. S9 MS/MS spectrum of -3 charged glycopeptide from trend line III in Fig. 9 for human
antithrom III in the paper. Underlined peak is precursor ion.
trap ce45-50V, 1mg/ml, 50/50 esi
1382.2
1060.4
100
1059.9
1381.9
%
1367.9
1382.9
1396.9
290.1
1367.2
1061.4
452.2
514.3
1397.9
979.4
1400.7
0
250
500
750
1000
1250
1500
1830.7
1750
m/z
Spectrum rough analysis:
Neutral loss of Hexose (Hex) includes 695.3-533.3; 1060.4-979.4; 1326.3-1164.4 and 1928.3-1906.3;
Neutral loss of N-acetylhexosamine (HexNAc) includes 736.4-533.3 and 823.4-602.3.
S-10