Mass spectrometry data confirming tetrameric α-synuclein N-terminal acetylation

Tetrameric α-synuclein (αS) is an elusive multimer of the dynamic neuronal protein implicated in Parkinson׳s disease. Through the data reported herein, we demonstrate that this high molecular weight multimer is N-acetylated. Coexpression of tetrameric αS in Escherichia coli with the NatB acetylase derived from yeast enables access to N-terminally acetylated αS (NAcαS), the native form in humans. Following purification and characterization as previously described by us in “Isolation of Recombinant Tetrameric N-acetylated α-synuclein” (Fernández and Lucas, 2018), the purified protein was excised from a native gel for confirmation of N-terminal acetylation. Through high-resolution mass spectrometry techniques, the identification of this helical tetramer as NAcαS has been clearly demonstrated.


How data was acquired
Mass spectrometry (Thermo Scientific LTQ Orbitrap Velos and Waters Synapt G2Si); Gel electrophoresis (BN-PAGE) Data  Fragmentation analysis following tryptic digestion of high molecular weight multimers preserves and verifies N-capping through in cellulo post-translational modification.
The data solidifies that recombinant platforms can be employed to generate human proteins with their native N-terminal acetylation in Escherichia coli. Fig. 1 shows a scheme that represents our strategy for mass spectrometry (MS) analysis. The cartoon signifies tetrameric NAc αS and its theoretical molecular weight. After co-expression and purification, the purified protein was run on a blue native PAGE (BN-PAGE) gel. Excised gel bands were analyzed by high resolution mass spectrometry via two separate methods. Data collected through Method 1 (diffusion of intact protein) is shown in Fig. 2 for the dissociated noncovalent NAc αS tetramer along with an inset of the theoretical isotopic distribution and relative abundance of an intact monomeric unit for comparison. Select data collected through Method 2 (trypsinization) is shown in Fig. 3 along with the associated peptide map of the N-terminal peptide as well as the theoretical b-/y-ion masses. Finally, Fig. 4 displays the quality of the mass data based on the low error and short retention times.

Protein expression and purification
Tetrameric NAc αS was expressed and purified as previously described [1,2]. Verification of the multimeric conformer was confirmed by BN-PAGE, and the associated native gel for the intact protein has been described in the related research article [1]. N-terminal acetylation was verified by mass spectrometry following gel electrophoresis as described herein (see Fig. 1 for synopsis).

Diffusion of intact protein from excised BN-PAGE gel (Method 1)
The 58 kD gel band corresponding to tetrameric NAc αS was excised from a BN-PAGE gel (Invitrogen) after destaining, and the purified protein was isolated through diffusion following band excision. The excised band was cut into small pieces and resuspended in ddH 2 O for 4 h [3]. Samples were then homogenized with a syringe, probe sonicated, and centrifuged at 14,500 g for 5 min. The supernatant was collected for MS analysis.

MS analysis of intact protein (Method 1)
The sample extracted from the 58 kD band of the tetrameric NAc αS sample was analyzed by direct infusion onto a Thermo Electron LTQ Orbitrap Velos [1]. Exact mass determination for one unit of the homotetrameric NAc αS was measured within less than 1 ppm of the theoretical mass (Fig. 2). Statistical analyses [4] of the isotopic distribution of the 17 þ charge envelope verified the identification of N-terminally acetylated αS. Theoretical mass isotopic distributions and abundances were calculated using the IsoPro 3.1 simulator, which is based on the Yergey algorithm [5].

Band excision and sample processing for trypsin digestion (Method 2)
The 58 kD band was excised from a BN-PAGE gel of the tetrameric NAc αS sample and divided into 1 mm segments to aid in digestion. Gel pieces were transferred to a siliconized tube and washed and destained in 200 mL 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 mL of 10 mM DTT in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the samples were alkylated in 30 mL 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces were dehydrated in 100 mL acetonitrile. The acetonitrile was removed and the gel pieces were rehydrated in 100 mL 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 mL acetonitrile, the acetonitrile removed and the pieces were completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/mL trypsin in 50 mM ammonium bicarbonate on ice for 10 min. Any excess trypsin solution was removed and 20 mL 50 mM ammonium bicarbonate added. The samples were digested overnight at 37°C, and the peptides formed from the digestion were extracted from the polyacrylamide in two 30 mL aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 mL for MS analysis. This protocol was also followed for the monomeric NAc αS gel band.

MS analysis of digested protein (Method 2)
Samples were analyzed by a Waters Synapt G2Si mass spectrometer system with a nanospray ion source interfaced to a Waters M-Class C18 reversed-phase capillary column. The peptides were injected onto the trap and analytical columns, and the peptides were eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.4 mL/min over 60 min. The nanospray ion source was operated at 3.5 kV. A lockspray compound was used to improve the mass accuracy of the analysis. The digests were analyzed using the double play capability of the instrument acquiring full scan mass spectra at low collision energy to determine peptide molecular weights and product ion spectra at high collision energy to determine amino acid sequence. This mode of analysis produces approximately 10,000 collision-induced dissociation (CID) spectra of ions ranging in abundance over several orders of magnitude. Not all CID spectra are derived from peptides. The data were analyzed by database searching using the ProteinLynx Global Server (PLGS, Waters Corp.) search algorithm against Uniprot's Human database. Fragmentation data was prepared for publication using Scaffold Q þ (Figs. 3 and 4). Theoretical b-/y-ion masses were determined using the ProteinProspector v.5.22.1, MS-Product, data mining program (Fig. 3B).
to thank Dr. Kristina T. Nelson, Director of the VCU Chemical and Proteomic Mass Spectrometry Core Facility, for her assistance with both data collection and interpretation.

Transparency document. Supporting information
Transparency data associated with this article can be found in the online version at https://doi.org/ 10.1016/j.dib.2018.09.026.