University of Groningen

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1 University of Groningen A method for site-specific labeling of multiple protein thiols Kuiper, Johanna M.; Pluta, Radek; Huibers, Wim H. C.; Fusetti, Fabrizia; Geertsma, Eric R.; Poolman, Bert Published in: Protein Science DOI: /pro.113 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2009 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Kuiper, J. M., Pluta, R., Huibers, W. H. C., Fusetti, F., Geertsma, E. R., & Poolman, B. (2009). A method for site-specific labeling of multiple protein thiols. Protein Science, 18(5), Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 B. Panel A: The amino acid sequence ofsulphate-binding protein (SBP) with and without signal sequence (SS) and after cleavage With tobacco etch virus (TEV) protease. Tile amino acid numbering for SBP Is given below the sequence at the top. Panel B: CoomaSSie Brilliam Blue (CBB)-stained 12.5% SDS-PAGE gels of SBP expressed with and without signal sequence. Tile numbers at tile right refer to the masses of the proteins in the marker lane. expressed in kda. Clearlv visible are the extra bands at higher masses for the protein expressed with SS. Expression of tile protein Without SS led to a single band In SDS-PAGE. Panel C: Expression ofsbp. E. coli MC 1061 comaimng pjmk I was cultivated IImil OD660=O.5. Protein expression was Induced by adding different amounts of 1.,(+)-arabinose (Ara). Samples were taken at time zero (sran of induction). t=45 min and t= 90 min. The cells wene harv'ested by cemrifugation and were heated to 100oC for 4 min and total cell lysates were analyzed by SDS-PAGE. Lane I: Marker proteins; The numbers at tile left refer to the masses In kda.: Lane 2: t=o & O.OOI%Ara; Lane 3: t=o & 0.01% Ara: Lane 4: t=0 & 0.1% Ara; Lane 5: t=45 & 0.001% Ara: Lane 6: t=45 & 0.01% Ara; Lane 7: t=45 & O.I%Ara: Lane 8: t= 90 & 0.001% Ara: Lane 9: t=90 & 0.01% Ara; Lane 10: t= 90 & 0.1% Ara. Clearly. IIpon induction with Ara. a protein with an apparem mass of ~38 kda becomes visible afler 45 and 90 min (see arrow).

3 Supplementary Imaterials: Kuiper et at (2008) A Imethodfor site-specific labelling of multiple protein thiols D. E. Expected Expected Observed M+ M2+ M12+ SBP w/o SS ,689 18,611 18,479 SBP w/o SS ,880 17,805 after TEV Panel D: Pan of the MALDI-Tof spectra of the wildtype SBP protein before and afler treatment wuh the TEl' protease. Left to the main peak of the Ml all extra peak at amu Is observed..iaflertreatment wuh TEV. the extra peak Is 110 longer observ'ed (lower pane/). Indicating some heterogeneity at tile C ter111nusof the protein. The data from the MS analysis are summarized III panel E. Panel E: Summary of MS data for SBP w & w/o signal sequence: M+ and Ml+ refer to tile singlv and doubly charged protein species.

4 F. Panels F: Ligand binding datafor Ihe determination of the KD of SBP for era42- and sa42-. Subpanel 1. To I ml of 1mM Tris- Hel. ph8.0. I mm EDTA was added 10 ill of 53 µm SBP. Subsequently. Cr042- from I mm stock solutions was added under contimuous stirring (.A). The control measurements were performed WIIh buffer ( ). Subpanel II. The difference III fluorescence between SBP and control sample was plotted against the t01a1concentration 01'0'0;-. Tile data was filled WIIh the general binding equation (Lanfermeijer el al., 1999). Subpanel III. To I ml of buffer was added 10 IlL of 53 11M SBP and I.j IlL of 100 mm CrO 42'.Subsequently, so42'was addedfrom 50 mm Stocks (.A). Also showii are Ihe measurements in which 1.5 µl of I M S042- was added at tile Start of the experiment ( ): these data were used 10 correct for bleaching effects. The KD for sa42- was calculated using lhe method of Kragh-Hansen (1983).

5 G. Panel G: The amino acid sequence or SBP without SS and the PoSitiOns or the cysteine mutations in the protein sequence. Panel H: SDS-(I1.5%) PAGE lunder non-reducing (Lane 1-4) and lunder reducing conditions (Lane 5-9). Mutant 3 migrates faster under non-reducing conditions due to Intramolecular disulfidelormation of Cys20 and Cys255. Tileproteins In lane 1-4 also show hands around 75 kda. which reflect initermolecular dimer.iormation. Tile numbers at tile lefl refer to tile masses of the marker In kda.

6 Supplementary Imaterials: Kuiper et at (2008) A Imethodfor site-specific labelling of multiple protein thiols Target residues Tryptic peptide Label Mass difference m/z sequence Cys 289 LFTIDEVFCGWAK none Cys 289 LFTIDEVFCGWAK NEM Cys 289 LFTIDEVFCGWAK OG Cys 20, Cys 23 ELYECYNCAFSAHWK none Cys 20, Cys 23 ELYECYNCAFSAHWK 2xNEM Cys 20, Cys 23 ELYECYNCAFSAHWK 2xOG Panel I: Sequences and m/z mluer; or cysteine-containing tryptic pep tides from tile RP I mutant of tile sulphate-binding protein (SB. functionalized wo or with NEM or Oregon Green (OG). Relevant mass spectra are shown 111 panel J.

7 no label NEM +NEM Panel J: Mass spectra of peptide LFTIDEVFCGWAK. unlabelled (upper panels). NEM-Iabelled (middle panels) and OG-Iabelled (lower panels); MS/MS spectrum o.f the NEM-labelled peptide Is shown In the bottom panel.