Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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linked products via affinity tags. 1,24,25 Identification of cross-linked<br />
peptide ions by CID tandem MS or MS n is not trivial as product<br />
ion spectra of these contain a number of product ions, i.e., band<br />
y-type ions, 26,27 originating from both peptides involved in<br />
the cross-linking product. To enable a more effective analysis of<br />
cross-linked peptides by CID tandem MS analysis, we have<br />
developed a novel concept for collision-induced dissociative crosslinking<br />
reagents. As such, the novel cross-linker contains a labile<br />
covalent bond located within the linker region, which is selective<br />
and preferably cleaved by collision activation in the gas phase.<br />
Highly efficient and reproducible cleavage of our novel urea-based<br />
cross-linker is mediated by a nucleophilic attack of a carbonyl<br />
oxygen in the cross-linker’s spacer chain at the urea carbonyl<br />
carbon. The synthesis and application of the predecessor of this<br />
cross-linker, a thiourea derivative, has been studied in detail<br />
previously. 14 However, that thiourea-based cross-linker exhibited<br />
a number of properties which were unfavorable for analyzing<br />
cross-linked products by CID in an automated fashion. Most<br />
importantly, we were unable to differentiate between the different<br />
types of cross-linked species, namely, peptides that are modified<br />
by a partially hydrolyzed cross-linker or intramolecular type 1<br />
cross-linked products.<br />
The urea-based cross-linker presented herein overcomes the<br />
deficiencies of the thiourea-based reagent. It leads to the formation<br />
of indicative fingerprint mass shifted product ions and neutral<br />
losses in the product ion mass spectra, which allow unambiguous<br />
identification of the different types of cross-linking products. The<br />
properties of the novel cross-linker were evaluated for the Munc<br />
13-1 peptide and the 28 kDa ligand binding domain of the<br />
peroxisome proliferator-activated receptor R (PPARR), impressively<br />
indicating its power for efficiently analyzing cross-linked<br />
products in a highly automated fashion.<br />
EXPERIMENTAL SECTION<br />
Materials. All chemicals and solvents used for synthesis of<br />
the cross-linker were used as purchased without further purification<br />
(Acros Organics, Geel, Belgium; ABCR, Karlsruhe, Germany).<br />
Dichloromethane and pyridine were dried by distillation from<br />
calcium hydride. All other solvents were distilled over a column<br />
prior to use.<br />
Buffer reagents and chemicals were obtained from Sigma<br />
(Taufkirchen, Germany). Proteases (trypsin and GluC, sequencing<br />
grade) were obtained from Roche Diagnostics (Mannheim,<br />
Germany). Nano-HPLC solvents were spectroscopic grade (Uvasol,<br />
VWR, Darmstadt, Germany). MALDI matrixes and calibration<br />
standards were purchased from Bruker Daltonik (Bremen,<br />
Germany). Water was purified with a Direct-Q5 water purification<br />
system (Millipore, Eschborn, Germany). The ligand binding<br />
domain of PPARR was expressed in Escherichia coli and purified<br />
(22) Schmidt, A.; Kalkhof, S.; Ihling, C.; Cooper, D. M.; Sinz, A. Eur. J. Mass<br />
Spectrom. 2005, 11, 525–534.<br />
(23) Taverner, T.; Hall, N. E.; O’Hair, R. A.; Simpson, R. J. J. Biol. Chem. 2002,<br />
277, 46487–46492.<br />
(24) Hurst, G. B.; Lankford, T. K.; Kennel, S. J. J. Am. Soc. Mass Spectrom. 2004,<br />
15, 832–839.<br />
(25) Sinz, A.; Kalkhof, S.; Ihling, C. J. Am. Soc. Mass Spectrom. 2005, 16, 1921–<br />
1931.<br />
(26) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601.<br />
(27) Papayannopoulos, I. A.; Biemann, K. Protein Sci. 1992, 1, 278–288.<br />
Scheme 1. Structure of the Symmetric<br />
NHS-BuUrBu-NHS Compound (1) for <strong>Chemical</strong><br />
Cross-Linking<br />
according to a previously published protocol. 28 The Munc13-1<br />
peptide was synthesized by Dr. O. Jahn (MPI for Experimental<br />
Medicine, Göttingen, Germany).<br />
Synthesis of the Cross-Linker. Aminobutanoic acid was<br />
dissolved in 4 M NaOH, and 0.175 equiv of triphosgen in dioxane<br />
was added at 0 °C (see Scheme S1 of the Supporting Information).<br />
The reaction mixture was brought to room temperature overnight,<br />
and the solid was removed by filtration. The solvent was removed<br />
under reduced pressure, and the residue was recrystallized from<br />
6 M hydrochloric acid. The product was isolated by filtration,<br />
washed with acetone, and dried in vacuum. HO-BuUrBu-OH was<br />
isolated as a colorless solid in a yield of 26%. 29 An abbreviated<br />
notation, BuUrBu, is used throughout this paper for the backbone<br />
of cross-linker 1, which is derived from urea (Ur) and aminobutyric<br />
acid (Bu) (Scheme 1).<br />
HO-BuUrBu-OH was dissolved in pyridine and treated with<br />
N-(trifluoroacetoxy)succinimidesprepared from N-hydroxysuccinimide<br />
(NHS) and trifluoroacetic anhydridesat 0 °C. 30 The<br />
reaction mixture was brought to room temperature within 2 h.<br />
After addition of ethyl acetate the raw product was isolated by<br />
filtration and dissolved in a mixture of dichloromethane and<br />
methanol. Insoluble components were removed by filtration, and<br />
the filtrate was evaporated. NHS-BuUrBu-NHS (1) was isolated<br />
as a colorless solid in a yield of 82%.<br />
Cross-Linking Reactions. For cross-linking experiments,<br />
aqueous stock solutions of Munc13-1 peptide (100 µg/mL) or<br />
PPARR ligand binding domain (2 mg/mL) were diluted with 20<br />
mM HEPES, 150 mM NaCl, 1 mM TCEP, 10% (v/v) glycerol, pH<br />
8.0, to a volume of 1 mL, giving a final protein/peptide concentration<br />
of 10 µM. The cross-linker (200 mM stock solution in DMSO)<br />
was added in 50, 100, and 200 M excess to the protein/peptide<br />
solution, and the reactions were allowed to proceed for 5, 15, 30,<br />
60, and 120 min. Reactions were quenched with ammonium<br />
bicarbonate (20 mM final concentration). One 200 µL aliquot was<br />
taken from each sample and stored at -20 °C before MS analysis.<br />
Cross-linked Munc13-1 peptide was digested with trypsin, whereas<br />
PPARR was digested either with trypsin or with trypsin/GluC<br />
(each 1:100 (w/w) enzyme to substrate ratio) overnight at 25 °C<br />
according to an existing protocol. 20 The resulting digests were<br />
stored at -20 °C before MS analysis.<br />
Linear Mode MALDI-TOF Mass Spectrometry. MALDI-<br />
TOF mass spectrometry of intact cross-linked PPARR was<br />
(28) Müller, M. Q.; Roth, C.; Sträter, N.; Sinz, A. Protein Expression Purif. 2008,<br />
62, 185–189.<br />
(29) Coe, S.; Kane, J. J.; Nguyen, T. L.; Toledo, L. M.; Wininger, E.; Fowler,<br />
F. W.; Lauher, J. W. J. Am. Chem. Soc. 1997, 119, 86–93.<br />
(30) Rao, T. S.; Nampalli, S.; Sekher, P.; Kumar, S. Tetrahedron Lett. 2002, 43,<br />
7793–7795.<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
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