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.

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.

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

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+

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+

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

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

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

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

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.

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.

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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