AI-64310-CRFT

High-Resolution, Accurate-Mass (HR/AM) and Intelligent Acquisition-Enabled Global Discovery and Quanti cation of Histones, Histone PTMS, and Histone

Modi cation Enzymes in Mesenchymal Stem Cells

lp drive targeted quantitative

ents were performed in an unbiased

cal samples to determine proteins

m the initial discovery results,

osen and further targeted discovery

bility of modified peptide

s. The combined results from the

pectral library consisting of

abundance distribution as well as

the discovery data was selected

ary information for the targeted

t and reference information to

real-time feedback facilitated

instrument duty cycle and detection

number of peptides quantified per

tudies on 36 proteins and 154

analogs (Table 1). The combined

tified modified peptides as well as

rrelated to somatic or stem cell

scence) (Figs 3-5).

Pictorial representation of

d peptide quantification using

tification, and real-time

r and product ion spectral

vity of data acquisition.

FIGURE 4. Differential

modification proteins

treatment with bleom

(cellular recovery afte

FIGURE 3. Differential

modification proteins

(2 hr treatment with bl

(cellular recovery afte

TABLE 1. Targeted proteins and peptides

0.00E+00 1.00E+08 2.00E+08 3.00E+08

HUMANHistoneH1.0

HUMANHistoneH3.1

HUMANHistoneH2B type 1-B

HUMANHistoneH2B type 1-A

HUMANHistoneH2B type 1-C/E/F/G/I

HUMANHistoneH2A type 2-B

HUMANHistoneH1.3

HUMANHistoneH1.2

HUMANHistoneH1x

HUMANHistoneH4

HUMAN Putative histoneH2B type 2-C

HUMANHistoneH2A type 1-D

HUMANHistoneH2A type 1-B/E

HUMANHistoneH1.5

HUMANHistoneH2A type 1-A

HUMANHistoneH2A.V

HUMAN Corehistonemacro-H2A.1

HUMAN Corehistonemacro-H2A.2

HUMANHistoneH3.1t

HUMANHistoneH1.4

HUMANHistoneH2B type 1-H

HUMANHistoneH1.1

HUMANHistone cluster 2 H3 pseudogene2

Axis

NoTreatment

Bleomycin

0.00E+00 5.00E+07 1

HUMANHistone-lysineN-methyltransferaseMLL3 (Fragment)

HUMANHistone deacetylase 5

HUMANHistone deacetylase 4

HUMANHistone-lysineN-methyltransferaseMLL2

HUMAN Keratin23 (Histone deacetylase inducible)

HUMANNHP2non-histone chromosome protein2-like 1 (S.

cerevisiae)

HUMANHistone acetyltransferase p300

HUMANHistone-binding proteinRBBP4

HUMANHistone deacetylase 1

HUMANHistone-lysineN-methyltransferase H3 lysine-79

specific

HUMANHistone deacetylase 2

HUMAN Probablehistone-lysine N-methyltransferase PRDM7

Histon

NoTreatment

Bleomyci

in)

Stop time for “watch list”

Spectral

Library

Experimental

Spectrum

Proteins

Peptide Sequence

>tr|H0Y765|H0Y765_HUMANHistone-lysineN-methyltransferaseMLL3 (Fragment)OS=Homo sapiensGN=MLL3PE=2SV=1

LM[Oxid]Q[Deamid]EVDR

>sp|P07305|H10_HUMANHistoneH1.0OS=Homo sapiensGN=H1F0PE=1SV=3

GVGASGSFR

YSDMIVAAIQAEK

>tr|Q5TEC6|Q5TEC6_HUMANHistone cluster2 H3 pseudogene2OS=Homo sapiensGN=HIST2H3PS2PE=1SV=1

EIAQEFK[Methyl]TDLR

EIAQEFKTDLR

>sp|Q92769|HDAC2_HUMANHistonedeacetylase2OS=Homo sapiensGN=HDAC2PE=1SV=2

LGC[Carboxymethyl]FNLTVK

>sp|Q86X55|CARM1_HUMANHistone-argininemethyltransferaseCARM1OS=Homo sapiensGN=CARM1PE=1SV=3

IVLDVGC[Carboxymethyl]GSGILSFFAAQ[Deamid]AGAR

>sp|Q9UQL6|HDAC5_HUMANHistonedeacetylase5OS=Homo sapiensGN=HDAC5PE=1SV=2

EKSK[di-Methyl]ESAIAS[Phosphoryl]TEVKLR

>sp|P68431|H31_HUMANHistoneH3.1OS=Homo sapiensGN=HIST1H3APE=1SV=2

K[Acet]QLATK[Acet]AAR

>sp|P33778|H2B1B_HUMANHistoneH2B type1-BOS=Homo sapiensGN=HIST1H2BBPE=1SV=2

KESYSIYVYK

IAGEASRLAHYNKR

ESYSIYVYK

AMGIMNSFVNDIFER

AMGIMNSFVNDIFER[Methyl]

AM[Oxid]GIMNSFVNDIFER

AMGIM[Oxid]NSFVNDIFER

AM[Oxid]GIM[Oxid]NSFVNDIFER

AM[Oxid]GIM[Oxid]NSFVNDIFER[Methyl]

STITSREIQ[Deamid]TAVRLLLPGELAK

AM[Oxid]GIMNS[S_42]FVNDIFER

AMGIM[Oxid]NS[S_42]FVNDIFER

A[A_42]M[Oxid]GIMNSFVNDIFER

A[A_42]MGIM[Oxid]NSFVNDIFER

AMGIM[Oxid]NS[S_42]FVN[Deamid]DIFER

AMGIM[Oxid]N[Deamid]S[S_42]FVNDIFER

AM[Oxid]GIMNS[S_42]FVNDIFER[Methyl]

AM[Oxid]GIM[Oxid]NS[S_42]FVNDIFER

A[A_42]M[Oxid]GIM[Oxid]NSFVNDIFER

A[A_42]M[Oxid]GIM[Oxid]NS[S_42]FVNDIFER

AMGIMN[Deamid]SFVNDIFER

AM[Oxid]GIMNSFVN[Deamid]DIFER

AMGIM[Oxid]NSFVN[Deamid]DIFER

AM[Oxid]GIMNSFVNDIFER[Methyl]

AM[Oxid]GIM[Oxid]N[Deamid]S[S_42]FVNDIFER

A[A_42]M[Oxid]GIM[Oxid]N[Deamid]SFVNDIFER

>sp|Q96A08|H2B1A_HUMANHistoneH2B type1-AOS=Homo sapiensGN=HIST1H2BAPE=1SV=3

HAVSEGTKAVTKYTSSK

LLLPGELAK[Methyl]

EIQTAVRLLLPGELAK

>sp|P62807|H2B1C_HUMANHistoneH2B type1-C/E/F/G/IOS=Homo sapiensGN=HIST1H2BCPE=1SV=4

KESYSVYVYK

SRKESYSVYVYK

KESYSVYVYKVLK

ESYSVYVYK

>sp|Q93079|H2B1H_HUMANHistoneH2B type1-HOS=Homo sapiensGN=HIST1H2BHPE=1SV=3

PDPA[A_42]KSAPAPK

>sp|Q8IUE6|H2A2B_HUMANHistoneH2A type2-BOS=Homo sapiensGN=HIST2H2ABPE=1SV=3

HLQLA[A_42]VRN[Deamid]DEELNK

>sp|P16402|H13_HUMANHistoneH1.3OS=Homo sapiensGN=HIST1H1DPE=1SV=2

SET[T_42]APLAPTIPAPAEK

S[S_42]ETAPLAPTIPAPAEK

>sp|Q9NQW5|PRDM7_HUMANProbablehistone-lysineN-methyltransferasePRDM7OS=Homo sapiensGN=PRDM7PE=2SV=2

LK[K_68]LELR

>sp|P16403|H12_HUMANHistoneH1.2OS=Homo sapiensGN=HIST1H1CPE=1SV=2

SGVSLAALK

KASGPPVSELITK

SGVSLAALKK

ASGPPVSELITK

S[S_42]ETAPAAPAAAPPAEK

>sp|Q02539|H11_HUMANHistoneH1.1OS=Homo sapiensGN=HIST1H1APE=1SV=3

K[Acet]PAKAAAAS[Phosphoryl]K[di-Methyl]K[Methyl]

ALAAAGYDVEK

>sp|P56524|HDAC4_HUMANHistonedeacetylase4OS=Homo sapiensGN=HDAC4PE=1SV=3

ALAHR[Methyl]NLN[Deamid]HC[Carboxymethyl]ISSDPR

>sp|O14686|MLL2_HUMANHistone-lysineN-methyltransferaseMLL2OS=Homo sapiensGN=MLL2PE=1SV=2

IIIIS[S_42]SR

>tr|Q8TC04|Q8TC04_HUMANKeratin23 (Histonedeacetylase inducible)OS=Homo sapiensGN=KRT23PE=2SV=1

LASYVEK

>tr|Q6FHM6|Q6FHM6_HUMANNHP2non-histone chromosomeprotein2-like1 (S. cerevisiae)OS=Homo sapiensGN=NHP2L1PE=2SV=1

AC[Carboxymethyl]GVSRPVIAC[Carboxymethyl]SVTIK

>sp|Q09472|EP300_HUMANHistoneacetyltransferasep300OS=Homo sapiensGN=EP300PE=1SV=2

TSKNK[K_68]SSLS[S_42]R

>sp|Q6DN03|H2B2C_HUMANPutativehistoneH2B type2-COS=Homo sapiensGN=HIST2H2BCPE=5SV=3

AM[Oxid]GIMNSFLNDIFER

AM[Oxid]GIMN[Deamid]SFLN[Deamid]DIFER

AM[Oxid]GIM[Oxid]NSFLNDIFER

>sp|Q92522|H1X_HUMANHistoneH1xOS=Homo sapiensGN=H1FXPE=1SV=1

YSQLVVETIR

>sp|P62805|H4_HUMANHistoneH4OS=Homo sapiensGN=HIST1H4APE=1SV=2

TVTAMDVVYALK

RKTVTAMDVVYALKR

RKTVTAM[Oxid]DVVYALKR

DNIQGITKPAIR

DNIQ[Deamid]GITKPAIR

DN[Deamid]IQGITKPAIR

DNIQGITKPAIR[Methyl]

DNIQGITKPAIRRLAR

TVTA[A_42]M[Oxid]DVVYALK

TVT[T_42]AM[Oxid]DVVYALK

GLGK[Acet]GGAK[Acet]R

GLGK[Acet]GGA[A_42]KR

DAVTYTEHAKRK

KTVTAMDVVYALK

KTVTAM[Oxid]DVVYALK

TVTAMDVVYALKR

TVTAMDVVYALK[Methyl]R

DNIQGITKPAIRR

TVTAM[Oxid]DVVYALKR

VFLEN[Deamid]VIR

KTVTA[A_42]M[Oxid]DVVYALK

KTVT[T_42]AM[Oxid]DVVYALK

KT[T_42]VTAM[Oxid]DVVYALK

K[Acet]TVTAM[Oxid]DVVYALK

VFLENVIR[Methyl]

TVTA[A_42]M[Oxid]DVVYALKR

VFLENVIR[tri-Methyl]

KTVTAMDVVYALKR

DAVTYTEHAK

ISGLIYEETR

ISGLIYEETR[Methyl]

ISGLIYEETR[tri-Methyl]

RISGLIYEETR

>sp|Q09028|RBBP4_HUMANHistone-bindingproteinRBBP4OS=Homo sapiensGN=RBBP4PE=1SV=3

TVALWDLR

>sp|P20671|H2A1D_HUMANHistoneH2A type1-DOS=Homo sapiensGN=HIST1H2ADPE=1SV=2

NDEELNKLLGK

>sp|P04908|H2A1B_HUMANHistoneH2A type1-B/EOS=Homo sapiensGN=HIST1H2ABPE=1SV=2

NDEELNKLLGR

VTIAQGGVLPNIQ[Deamid]AVLLPK

VTIAQGGVLPN[Deamid]IQAVLLPK

VTIAQ[Deamid]GGVLPNIQAVLLPK

VTIAQGGVLPN[Deamid]IQ[Deamid]AVLLPK

VTIAQ[Deamid]GGVLPNIQ[Deamid]AVLLPK

VTIAQGGVLPNIQ[Deamid]AVLLPK[Methyl]

VTIAQGGVLPNIQ[Deamid]AVLLPKK

VTIAQGGVLPN[Deamid]IQAVLLPKK

VTIAQ[Deamid]GGVLPNIQAVLLPKK

VTIAQGGVLPN[Deamid]IQAVLLPK[Methyl]

>sp|P16401|H15_HUMANHistoneH1.5OS=Homo sapiensGN=HIST1H1BPE=1SV=3

KATGPPVSELITK

NGLSLAALKK

ALAAGGYDVEK

ATGPPVSELITK

>sp|Q13547|HDAC1_HUMANHistonedeacetylase1OS=Homo sapiensGN=HDAC1PE=1SV=1

MTHNLLLNYGLYR

YGEYFPGTGDLR

>sp|Q8TEK3|DOT1L_HUMANHistone-lysineN-methyltransferase H3 lysine-79specificOS=Homo sapiensGN=DOT1LPE=1SV=2

QLDGLAGLK[tri-Methyl]GEGSR

>sp|Q96QV6|H2A1A_HUMANHistoneH2A type1-AOS=Homo sapiensGN=HIST1H2AAPE=1SV=3

LLRKGNYAER

SSRAGLQFPVGR

HLQLAIR

HLQ[Deamid]LAIR

AGLQFPVGR

AGLQFPVGR[Methyl]

HLQLAIRN[Deamid]DEELNK

LLGGVTIAQGGVLPNIQ[Deamid]AVLLPK

LLGGVTIAQGGVLPN[Deamid]IQAVLLPK

LLGGVTIAQ[Deamid]GGVLPNIQAVLLPK

>sp|Q71UI9|H2AV_HUMANHistoneH2A.VOS=Homo sapiensGN=H2AFVPE=1SV=3

ATIAGGGVIPHIHK

>sp|O75367|H2AY_HUMANCorehistonemacro-H2A.1OS=Homo sapiensGN=H2AFYPE=1SV=4

NC[Carboxymethyl]LALADDK

SIAFPSIGSGR

NC[Carboxymethyl]LALADDKK

LEAIITPPPAK

EFVEAVLELR

NGPLEVAGAAVSAGHGLPAK

GKLEAIITPPPAK

AISSYFVSTMSSSIK

>sp|Q9P0M6|H2AW_HUMANCorehistonemacro-H2A.2OS=Homo sapiensGN=H2AFY2PE=1SV=3

AGVIFPVGR

>sp|Q16695|H31T_HUMANHistoneH3.1tOS=Homo sapiensGN=HIST3H3PE=1SV=3

STELLIRKLPFQR

STELLIR

YQKSTELLIR

STELLIR[Methyl]

EIAQDFK

EIAQ[Deamid]DFK

EIAQDFK[Methyl]TDLR

EIAQ[Deamid]DFK[Methyl]TDLR

EIAQDFK[di-Methyl]TDLR

VTIM[Oxid]PKDIQLAR

RYQKSTELLIR

KS[S_42]APATGGVK

K[Acet]SAPATGGVK

K[K_56]SAPATGGVK

K[Methyl]S[S_42]APATGGVK

YRPGTVALR

>sp|P10412|H14_HUMANHistoneH1.4OS=Homo sapiensGN=HIST1H1EPE=1SV=2

K[Acet]ATGAATPK[tri-Methyl]K[di-Methyl]

S[S_42]ETAPAAPAAPAPAEK

ment ions from KESYSIYVKYK

ecursor ion charge state 2

otope

e isotope

0.00E+00

1.00E+09

HUMANHistoneH1.0

HUMANHistoneH3.1

HUMANHistoneH2B type 1-B

HUMANHistoneH2B type 1-A

HUMANHistoneH2B type 1-C/E/F/G/I

HUMANHistoneH2A type 2-B

HUMANHistoneH1.3

HUMANHistoneH1.2

HUMANHistoneH1x

HUMANHistoneH4

HUMAN Putative histoneH2B type 2-C

HUMANHistoneH2A type 1-D

HUMANHistoneH2A type 1-B/E

HUMANHistoneH1.5

HUMANHistoneH2A type 1-A

HUMANHistoneH2A.V

HUMAN Corehistonemacro-H2A.1

HUMAN Corehistonemacro-H2A.2

HUMANHistoneH3.1t

HUMANHistoneH1.4

HUMANHistoneH2B type 1-H

HUMANHistoneH1.1

HUMANHistone cluster 2 H3 pseudogene2

NoTreatment

Bleomycin

0.00E+00

4.00E

HUMANHistone-lysineN-methyltransferaseMLL3 (Fragment)

HUMANHistone deacetylase 5

HUMANHistone deacetylase 4

HUMANHistone-lysineN-methyltransferaseMLL2

HUMAN Keratin23 (Histone deacetylase inducible)

HUMANNHP2non-histone chromosome protein2-like 1 (S.

cerevisiae)

HUMANHistone acetyltransferase p300

HUMANHistone-binding proteinRBBP4

HUMANHistone deacetylase 1

HUMANHistone-lysineN-methyltransferase H3 lysine-79 specific

HUMANHistone deacetylase 2

HUMAN Probablehistone-lysine N-methyltransferase PRDM7

Histon

NoTreatment

Bleomy