20mm glcnac injection conductance...2009/05/19 · mh 3 2+-2hexnac mh 3 2+-hexnac z15 z16 z14 z13...
TRANSCRIPT
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0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
Sample Injection 20mM GlcNAc Injection
GlcNAc-modifiedpeptide elution
Complex glycanelution
ConductanceUV Abs: 215nm
UV Chromatogram of LWAC separation of mouse PSD tryptic peptides.
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The following pages contain annotated spectra supporting the O-GlcNAc site identifications determined in this study. Peptide sequence and site assignments are displayed at the top of the page, then spectra with peak assignments are below. These assignments are based on search engine results, where no attempt was made to distinguish between z. and z+1 ions.
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m/z 614.318 3+VT(HexNAc)QHFAKEPQDPLK – Protein Bassoon
Modified c2 identifies site as residue 1354
z4
c2c3
z5
c4
z7
z8z9
c7z10 z11
c10
z12
-
m/z 829.050 3+EVGMTFSQGPGS(HexNAc)PATT(HexNAc)ASPTR – Protein BassoonSingly modified c15 and unmodified z4 identifies site as residue1395
Other modified residue is either 1391 or 1394
z2
z4c4 c5
c6
c7
c8
MH
32+ -
2Hex
NA
c
MH
32+ -
Hex
NA
cz16z15
z14z13
z10
c15
-
m/z 934.439 5+SPSTS(HexNAc)STIHSYGQPPTTANYGSQTEELPHAPS(HexNAc)GPPGSGRAPR
– Protein BassoonMass difference between c4-c5 identifies site as residue1418
z3c3 c4z5 b102+
c5b132+
c9
z363+
z403+
z393+
z372+
-
m/z 893.82 5+SPSTSSTIHSYGQPPTTANYGSQTEELPHAPS(HexNAc)GPPGSGRAPR
– Protein BassoonUnmodified z6 and modified z11 identifies site as residue 1445
c3z3
c4c5
z6c6
c8
c7
z333+
z343+z11
z363+
z403+
z393+z404+
z302+ z322+
-
m/z 775.718 3+ATAEFSTQTPSLT(HexNAc)LSSDIPR – Protein Bassoon
Unmodified z7 and modified z9 identify site as residue 1517
c5
z3
z5
c7y4
y5 z7
MH
22+ -
Hex
NA
c
z12 z15c15 z16
c17c14
y6y10
z9
-
m/z 647.320 4+SPGPPSPMVAQGT(HexNAc)QTPHRPSTPR – Protein Bassoon
Modified c14 and z12 identifies site as residue 1537
z72+
z3
z92+
c5c7
z6
z122+
c172+
c222+
z12 c19c14
c16
-
c5
z2 z3z4
m/z 738.392 4+ISSVPGTS(HexNAc)RVEPGPRPPGT(HexNAc)AVVDLR – Protein Bassoon
Mass difference between c6-c7 identifies one site as residue 1657Modified z11 identifies other site as residue 1666
z5
c6z6
z112+ z152+ c9
c7z172+
z182+ c222+z192+
z15
c192+c242+
z13
-
m/z 493.600 3+KYGLALDPVS(HexNAc)GR – Protein Bassoon
Modified z3 identifies residue 1707
z3b6 z6
z7
z9z8 z10c10
c11
z11
-
z9z4 z5
z16
z13
y3 y4 y5b6
y11*
y11y9
y13
y13*b14*
b16*
MH
+ -H
exN
Ac
m/z 983.481 2+LDFGQGSGS(HexNAc)PVC(Carbamidomethyl)LAQVK – Protein Bassoon
z9 in ETD identifies site as residue 1772
CID
ETD
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m/z 665.585 4+T(HexNAc)QGVVGPGPHEEQRPYPQGLPGR – Protein Bassoon
Modified c3 identifies site as residue 1962
z4
z5c3
z102+
z112+
z122+
z132+
c192+ c13c15c12
c222+
-
m/z 706.823 2+HS(HexNAc)YSLGFADGR – Protein Bassoon
Mass difference between z9-z10 identifies site as residue 2027
z4
z9 c8z5z6
z7z8
c10
z10
-
z4z3
z5
z6
m/z 706.824 2+HSYS(HexNAc)LGFADGR – Protein Bassoon
z7-z8 identifies residue 2029
z7
z8
c8 z9
c10
-
m/z 464.509 4+HPTDLLS(HexNAc)HPLPLRR – Protein Bassoon
Mass difference between c6-c7 identifies site as residue 2058
z1
z52+c3
z3
c6
c132+ c7
c9
z10z9
-
m/z 544.248 3+YS(HexNAc)SVSNIYSDHR – Protein Bassoon
Unmodified z10 and modified c2 identify residue 2067
z2 z3z4
z5
c6
z7c7 c8 c9
c10c11c5c4
c2
z10
-
544.248 3+YSS(HexNAc)VSNIYSDHR – Protein Bassoon
Modified c4 and z10 identify site as residue 2068
z2z3 z4
z5c6
z7
c7
c8c9 c10
z10
c11
c5c4
-
m/z 544.249 3+YSSVS(HexNAc)NIYSDHR – Protein Bassoon
Mass difference between z7-z8 identifies site as residue 2070
z2c3
z3z4
c4
z92+
z5
z102+c5
c6
z7
c7
c8
z8c9 c10
c11
-
z6
m/z 907.104 3+YGPRGDAVGFQEAS(HexNAc)LAQYSATTAR – Protein Bassoon
Modified c14 identifies site as residue 2091
z4 b6c142+
z12 z13c14
z14
c15z15
z16c10
MH
32+ -
Hex
NA
c
-
c3
c4
m/z 821.163 4+HGSGSGGPDLVQYQPQHGPGLS(HexNAc)APQGLAPLR – Protein Bassoon
Unmodified c10 identifies site as residue 2141
z4
c6
z5
z6
c8c9
c10
c222+c242+
c272+c252+
z222+z292+
z272+
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m/z 644.857 2+QLLPS(HexNAc)TATVR – Protein Bassoon
Mixture of peptides modified on two different residues:Unmodified z5 identifies one site as residue 2188
z3
z4
z5z5*
z6y7
z8
z9
-
m/z 621.661 3+AS(HexNAc)GAGGPPRPELPAGVAR – Protein Bassoon
Residue 2295 is the only potential modification site
z3z4
z5z7
z152+
c11
z13
c13z15
c14
c15 c17
-
z6
m/z 898.445 2+EEPFST(HexNAc)T(HexNAc)APAVIK – Protein Bassoon
Mass difference between z6-z7 identifies one site as residue 2318Mass difference between z7-z8 identifies other site as residue 2317
z7
z9
c9
z10c11
MH
+ -H
exN
Ac
z12
c12
z8
-
m/z 541.957 3+VS(HexNAc)PAIHITAATDPK – Protein Bassoon
Modified c4 identifies residue 2694
y2z3
z4
z5
z6
z7c11
b12c9 z11
z9
c4
-
z3
m/z 606.580 3+VSPAIHIT(HexNAc)AAT(HexNAc)DPK – Protein Bassoon
Modified z5 and doubly modified z9 identify sites as residues 2700 and 2703
c5 z5
z4
c9c8 z9
c11z11
c6
-
c11
m/z 687.046 3+GLAGPTT(HexNAc)VPAT(HexNAc)KASLLR - Protein bassoon
Mass difference between z10-z11 identifies one site as residue 2941.Mass difference between c10-c11 identifies other site as residue 2945.
z6
z5z4
z3z2 z10c10
z8c13
c14
z12
z11 c16
-
z2
m/z 588.986 3+TIPKS(HexNAc)EVKVT(HexNAc)EK – Protein Piccolo
Mass difference between c4-c5 identifies one site as residue 2634Mass difference between z2-z3 identifies other site as residue 2639
c4z3
z4
c5z7
c7 c8c9
z8z9
c11
c10 z11
z112+c112+
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m/z 480.905 3+QT(HexNAc)TANEVYRR – Protein PiccoloModified c2 identifies site as residue 2656
z2
c2 c3z4z6
c6
z7z8
c8 c9
-
z8z10c7
z9 c10
c11z11
z5
z7
MH
+ -H
exN
Ac
m/z 753.337 2+VS(HexNAc)TGEVMDYSSK - Protein piccolo
Mass difference between z10-z11 identifies site as residue 2930
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m/z 990.500 3+TTGPYPET(HexNAc)RQVISGVGISTPQYSTAR – Protein Piccolo
Modified b11 and y21 identify site as residue 2948
y11y212+*
y7 y8
y9b152+b4b15 b17 b18
y242+
b11*
CID
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m/z 642.836 2+IT(HexNAc)STYEVIR - Protein piccolo
Mass difference between z7-z8 identifies site as residue 3873
z4
z3z2
z5
z6 z7
z8
MH
+ -H
exN
Ac
-
m/z 620.629 3+RASSPGYIDSPTYS(HexNAc)R – Actin-binding LIM protein 3
Unmodified c12 identifies site as residue 383
c6c3
c5
c9 z9z8
c12
MH
2+-H
exN
Ac
-
z4
m/z 685.329 2+SS(HexNAc)TPTSYQAPK – Actin-binding LIM protein 3
Mass difference between z9-z10 identifies site as residue 419
z5
z9
c8
c10
z10
MH
+ -H
exN
Ac
-
m/z 685.329 2+SSTPTS(HexNAc)YQAPK – Actin-binding LIM protein 3Mass difference between z5-z6 identifies residue 423
z4
z5
z6
z7z9
c8
c10
-
m/z 724.666 3+SSSYADPWTPPRS(HexNAc)STSSR - Actin-binding LIM protein 3
Mass difference between c12-c13 identifies residue 546
z3z2
z4
z5z6
z132+
z142+
z152+z162+
z10
z11c12c13
c14 c15
z15
-
m/z 685.329 2+SS(HexNAc)TPTSYQAPK - Actin-binding LIM protein 3
Mass difference between z9-z10 identifies site as residue 547
z6
z5
z4z9
c8 z10
c10
MH
+ -H
exN
Ac
-
m/z 783.854 2+S(HexNAc)SSYADPWTPPR - Actin-binding LIM protein 3
Unmodified z11 identifies site as residue 534
z4
z7
z8z9
z10
c11z11
MH
+ -H
exN
Ac
y6
-
m/z 843.402 2+STS(HexNAc)QGSINSPVYSR - Actin-binding LIM protein 1
Mass difference between z11-z12 identifies site as residue 496
z4z6 z8 z10
z9
z11
z12
z13z7
-
z6z7 z9
z13
z12
z11 c13
m/z 843.402 2+STSQGS(HexNAc)INSPVYSR - Actin-binding LIM protein 1Unmodified z7 and modified z9 identify site as residue 499
-
c15
c14c12
z10
c11
z8z7
z6z132+
z112+
z4
z3
z152+
z2
m/z 638.965 3+VSGSPSSGFRS(HexNAc)QSWSR - Neurofilament medium polypeptide
Unmodified z4, but modified z6 identifies site as residue 37
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m/z 486.903 3+GSPS(HexNAc)TVSSSYKR - Neurofilament medium polypeptide
Mass difference between z8-z9 identifies site as residue 46
c10y11c11c9
c8
c7z2 z3z4
z8
z112+
z9
-
m/z 726.121 4+FSTFSGSITGPLYTHRQPSVT(HexNAc)ISSK - Neurofilament medium polypeptide
Mass difference between c20-c21 identifies site as residue 430
z4
z3
z102+
z122+c182+
z212+
c212+
c192+c202+
z192+
z232+z222+
c15z13
-
m/z 975.965 2+SAYSSYSAPVS(HexNAc)SSLSVR - Neurofilament light polypeptide
Mass difference between z6-z7 identifies site as residue 48
z6z5
z7z10
z11
y9 y11z13
z14
z12
MH+-HexNAc
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m/z 833.406 3+LSFTSVGSITSGYS(HexNAc)QSSQVFGR - Neurofilament light polypeptide
Mass difference between c13-c14 identifies site as residue 414
z4z3
z5
z6z7
MH22+-HexNAcc15
c14
c17
c13
z10
-
m/z 953.966 2+S(HexNAc)GSLSSSPSNTPSASPLK - 270 kDa ankyrin G isoform
Unmodified z17 identifies site as residue 1520
z12z13
z14
c13c14
z15
z16
z17
c17z9
z8 z10
-
m/z 592.605 3+S(Phospho)SSPPRT(HexNAc)T(HexNAc)TTVR - Disks large-associated protein 1
Mass difference between z4-z5 identifies site as residue 525Mass difference between z5-z6 identifies site as residue 526
One of the serines is phosphorylated.
z1
z3z4
c5
c6
z5
c7
z6
c8
c9
c10
c11
-
m/z 703.392 2+TT(HexNAc)SGSIITVVPK – Protein EMSY
Mass difference between z10-z11 identifies site as residue 499
z4z5
z6z7
z8 z10
c8 c9 z11
c11
MH
+ -H
exN
Ac
-
m/z 733.694 3+SLSQSQGDPLPPAHT(HexNAc)GTFR - Catenin delta-2
Unmodified c14 and modified c16 identify site as residue 447
z2 z3
z4
c5c7
z152+
c9z172+
c14 z14 c16z182+
c17
-
m/z 951.475 2+TS(HexNAc)TAPSSPGVDSVPLQR - Catenin delta-2
Mass difference between z15-z16 identifies site as residue 453
z5z6 z8 z14 z15
z16
z11c16
-
y10
m/z 534.930 3+TPFHTSLHSGTS(HexNAc)K – DematinModified z2 identifies site as residue 285
z2
c4
z3
c5
z5 c122+
z7
c8
c11z9
c10
z8
c12
-
m/z 542.279 3+RTPVS(HexNAc)YQNTISR - Nuclear receptor corepressor 1Mass difference between c4-c5 identifies site as residue 1496
z4
c3
z3
c4
z5z6 c6
c7c8 z9
c10
c11
c5
-
m/z 583.280 3+SAGQT(HexNAc)QSLTIC(Carbamidomethyl)HNK - Polyhomeotic-like protein 3
Mass difference between c4-c5 identifies site as residue 238
z3c4
c5z5 c8
z8 z9 c10
c11
c12
-
m/z 673.296 3+EQTYPC(Carbamidomethyl)YS(HexNAc)GTSGLHSK - Human immunodeficiency
virus type I enhancer-binding protein 2Unmodified z6 and modified z9 identify site as residue 1271
z2z3
c3 z4
z6z5
c6
z9
z10 c10c12
z13
c13 c14
c11
-
z14
c14
z10z13c5
c4c3c6 z16z5
c7
m/z 697.325 3+RASQSSLESSTGPSYS(HexNAc)RS - Regulating synaptic membrane exocytosis
protein 2Unmodified c14 and modified c17 identify site as residue 1528
c172+
-
The following pages contain annotated spectra supporting the N-GlcNAc site identifications determined in this study. Peptide sequence and site assignments are displayed at the top of the page, then spectra with peak assignments are below. These assignments are based on search engine results, where no attempt was made to distinguish between z. and z+1 ions.
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z3z4
z5z6 z8
z9
z122+
z11c9c7
c4c3
c5
c10c11
c12
m/z 594.946 3+IQDFN(HexNAc)YTDHTLGR – Gamma-aminobutyric acid type B receptor subunit 2
Mass difference between c4 to c5 identifies asparagine 388 as GlcNAc modification site.
-
z3z6 y7 z9
z10c10
z8z4
z5
m/z 684.322 2+FGTVPN(HexNAc)GSTER - Glutamate [NMDA] receptor subunit epsilon-2 precursor
Mass difference between z5 and z6 identifies asparagine 688 as GlcNAc modification site.
-
z4
z6
c5
z10c7
c9c10
z11z12
c12
z13
c13
c14c15
664.289 2+EIC(Carbamidomethyl)SGN(HexNAc)SSQC(Carbamidomethyl)APNVHK – ADAM 22
Mass difference between z10-z11 identifies asparagine 517 as GlcNAc modification site.
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m/z 742.733 3+KLVQVGIYN(HexNAc)GTHVIPNDR – Glutamate [NMDA] receptor subunit zeta-1 precursor
Unmodified c8 but modified c10 identify asparagine 368 as GlcNAc modification site.
c4z5
c5 c6
c112+c3
c8 z172+ z10z12
z14c13c11
c10
-
m/z 545.649 3+HRAN(HexNAc)ATLLLGPLR - Neurocan core protein precursor
Mass difference between c4-c5 identifies asparagine 121 as GlcNAc modification site
z4
c3c4
z5
c5
c7
c9
z10
z11
c12z6
-
m/z 503.000 4+WSC(Carbamidomethyl)DHKQN(HexNAc)ITYLLK – Oligodendrocyte-myelin glycoprotein
precursorMass difference between z6-z7 identifies asparagine 234 as GlcNAc modification site.
z3 c3z2
c62+
c72+
c4
z102+z112+
c112+
c122+
c132+z7
z8
z9c8z10
c10
z11
b122+
b12
z6
c5c92+
-
z4
m/z 616.340 3+KFHVN(HexNAc)YTQPLVAVK - Sodium/potassium-transporting ATPase subunit beta-2
Mass difference between z9-z10 identifies asparagine 238 as GlcNAc modification site.
c4
z5
z7
c7
z9z10
c9
c10z11
c11 c13c12
-
m/z 594.031 4+HEN(HexNAc)NTKDNSIQHEFSLTR - Thy-1 membrane glycoprotein precursor
Unmodified c2 and z15 identify asparagine 42 as GlcNAc modification site.
c2
z2 z3z4
c3 orz102+
c92+
z112+
c102+
c4 orz122+
c122+c112+
c132+c142+
c6
c152+
z162+z142+
c162+
c172+ z10
z11 z12c11
z13c12 z15 c14
c13
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