Synthetic Peptide Reference Standards for PTM Proteomics Karsten Schnatbaum1, Daniel P. Zolg2, Mathias Wilhelm2, Tobias Knaute1, Johannes Zerweck1,

Guido Morbach1, Lars Hornberger1, Holger Wenschuh1, Bernhard Kuster2,3,4, Ulf Reimer1* 1 JPT Peptide Technologies GmbH, Berlin, Germany, 2 Chair of Proteomics and Bioanalytics, Technical University of Munich, Freising, Germany, 3 Center for Integrated Protein Science Munich, Freising, Germany, 4 Bavarian Center for Biomolecular Mass Spectrometry, Freising, Germany.

* Correspondence should be addressed to Karsten Schnatbaum (schnatbaum@jpt.com)

IntroductionDespite recent improvements, PTM proteomics is stillchallenging.1 Typical problems are a) low endogenousabundance, b) low ionization intensity, c) changedfragmentation, d) limited stability during proteomicsworkflows, and/or e) complex fragmentation spectrainterpretation, especially regarding correct PTM sitelocalization.Defined synthetic PTM reference peptides support PTMproteomics and help to overcome challenges.Accordingly, the recently launched ProteomeToolsproject2 will incorporate reference spectra for hundredthousands of PTM modified peptides. The goal of thecurrent study is the development of defined sets of PTMreference standards that should be valuable tools foroptimizing and standardizing proteomics workflows.

Methods and ResultsTwo sets of PTM reference peptides were synthesizedin a stable-isotope labeled (SIL) form and analyzed byLC-MS/MS (Figure 1, Tables 1 and 2). Peptides for set Awere non-quantified, while peptides for set B werepurified to high purity and absolutely quantified.

Figure 1: Overview of PTMs included in the study.

The peptide sequences were chosen from available MSdatasets or – when this was not possible – taken fromUniprot. The PTMs were incorporated by usingrespective pre-synthesized modified amino acid buildingblocks for SPOT3 (set A) or classical resin-basedsynthesis (set B). LC-MS/MS analysis was done on aFusion Lumos instrument, data analysis with MaxQuant.

Figure 3: a) Determined MaxQuant Andromeda scores for allpeptides from final set A. b) Determined charge distribution forthree selected Lys modifications.

We are currently working on the use of the peptide setsfor optimizing MS conditions and on the analysis ofretention time shifts.To support the PTM proteomics community it is plannedto make both peptide sets commercially available asSIL labelled peptides. Customized sets can besynthesized quickly at reasonable costs with the high-throughput SPOT peptide synthesis platform. Thesepeptides sets can be used as reference standards or foranswering specific questions.

Conclusion

References(1) (a) Olsen, J. V. et al. Mol. Cell. Proteomics 2013, 12, 3444-2452. (b) Doll, S. et al. ACS Chem Biol. 2015, 10(1), 63-71.(2) Zolg, D. P. et al. Nat. Methods 2017, 14(3), 259-262.(3) Wenschuh, H., et al.. Biopolymers 2000, 55, 188-206.

Tables 1 and 2: Synthesized peptide sets.

For the development of set A, 170 or more peptideswere synthesized for each PTM in non-isotope labelledform. It turned out (Figure 2) that the recovery for allpeptides in LC-MS/MS experiments was very high. Thisencompassed not only frequently analyzed PTMs likeLys(Ac) (in fact 49,586 previously reported Lys(Ac)modified proteotypic peptides were synthesized), butalso less frequently analyzed PTMs like crotonylated orhydroxyisobutyrylated Lys.

Figure 2: Recovery (proportion of successfully detected peptides)for PTM modified peptides to be included in set A. The number ofsynthesized peptides is shown in brackets below the bars.

Based on the obtained data the optimal peptides for setA were selected (100 peptides per PTM). All analyzedpeptides showed very high andromeda scores (average>100 in all cases, Figure 3a). Although still high, thescores for some PTMs were somewhat lower. Thesecan be explained by lower synthetic accessibility (i.e.Lys Malonyl) or a shift of the preferred ionization statefrom 2+ to 3+ (e.g. Lys Acetyl to Lys Methyl, Figure 3b).

Synthetic peptide reference standards wereprepared as tools for optimizing andstandardizing proteomics workflows andanalysis conditions.The catalogue of modifications can easily beextended as new modifications aredescribed.

Final Peptide Set A: Non-quantified peptides

Name # PeptidesLys Unmodified 100Lys(Methyl) 100Lys(Dimethyl) 100Lys(Trimethyl) 100Lys(Formyl) 100Lys(Acetyl) 100Lys(Propionyl) 100Lys(Butyryl) 100Lys(Malonyl) 100Lys(Succinyl) 100Lys(Glutaryl) 100Lys(Crotonyl) 100Lys(Hydroxyisobutyryl) 100Lys(Biotinyl) 100Lys(GG) (Ubiquitinyl) 100Arg Unmodified 100Arg(Methyl) 100Arg(Dimethyl asymm) 100Arg(Dimethyl symm) 100Cit 100Nitrotyr Unmodified 100Nitrotyr 100Hyp Unmodified 100Hyp 100Glycosyl Unmodified 100Glycosyl Ser/Thr(alphaDGalNAc) 100Glycosyl Ser/Thr(betaDGlcNAc) 100

∑ 2700

Final Peptide Set B: Absolutely quantified peptides

Name # PeptidesLys(Methyl) 6Lys(Dimethyl) 6Lys(Trimethyl) 6Lys(Formyl) 6Lys(Acetyl) 6Lys(Propionyl) 6Lys(Butyryl) 6Lys(Malonyl) 6Lys(Succinyl) 6Lys(Glutaryl) 6Lys(Crotonyl) 6Lys(Hydroxyisobutyryl) 6Lys(Biotinyl) 6Lys(GG) (Ubiquitinyl) 6Arg(Methyl) 6Arg(Dimethyl asymm) 6Arg(Dimethyl symm) 6Phospho Unmodified 4Phospho-Ser 5Phospho-Thr 5Phospho-Tyr 4Tyr Sulfatation Unmodified 5Tyr Sulfatation 6Glycosyl Unmodified 3Glycosyl Ser(alphaDGalNAc) 4Glycosyl Thr(alphaDGalNAc) 3Glycosyl Asn(betaDGlcNAc) 1

∑ 142

OCH

NHCH2

CH2

CH2

HN

NH HN

NH

H2N O

N

N

N

NH

NHHN

Citrulline

Dimethylsymmetric

Dimethylasymmetric

Monomethyl

Arg

OCH

NHCH2

CH2

CH2

CH2

Lys

HN

NHHN

N

N

HN

NHNH

NH

NH

O

O OO

O OHO

OOH

O

Monomethyl

Dimethyl

Trimethyl

Formyl

Acetyl PropionylButyryl

Malonyl

Succinyl

Glutaryl

HN

NH

NH

NH

O

O OH

O

Crotonyl

Hydroxyisobutyryl

Biotinyl

Ubiquitinyl(digested)

OO

OH

O

NH NH2

OS

NH

HN OH

H

OCH

NHCH2

OPOH

O

TyrO

CH

NHCHOPOH

Ser/Thr

Me/H

O OH

OCH

NHCH2

OSOH

OO

Tyr

OH

OHO

AcNH

OHOH

OCH

NHCHO

Ser/Thr

Me/H

OCH

NHCH2

Asn

OHOHO

NHAc

HN

OH

O

OCH

NHCH2

OH

Tyr

NO2

Phospho Phospho Sulfo Nitro

alphaDGalNAc

betaDGlcNAc

a)

b)