Comprehensive identification of protein disulfide bonds with pepsin/trypsin digestion, Orbitrap HCD and Spectrum Identification Machine

Abstract

Disulfide bonds (SS) are post-translational modifications important for the proper folding and stabilization of many cellular proteins with therapeutic uses, including antibodies and other biologics. With budding advances of biologics and biosimilars, there is a mounting need for a robust method for accurate identification of SS. Even though several mass spectrometry methods have emerged for this task, their practical use rests on the broad effectiveness of both sample preparation methods and bioinformatics tools. Here we present a new protocol tailored toward mapping SS; it uses readily available reagents, instruments, and software. For sample preparation, a 4-h pepsin digestion at pH 1.3 followed by an overnight trypsin digestion at pH 6.5 can maximize the release of SS-containing peptides from non-reduced proteins, while minimizing SS scrambling. For LC/MS/MS analysis, SS-containing peptides can be efficiently fragmented with HCD in a Q Exactive Orbitrap mass spectrometer, preserving SS for subsequent identification. Our bioinformatics protocol describes how we tailored our freely downloadable and easy-to-use software, Spectrum Identification Machine for Cross-Linked Peptides (SIM-XL), to minimize false identification and facilitate manual validation of SS-peptide mass spectra. To substantiate this optimized method, we've comprehensively identified 14 out of 17 known SS in BSA. SIGNIFICANCE: Comprehensive and accurate identification of SS in proteins is critical for elucidating protein structures and functions. Yet, it is far from routine to accomplish this task in many analytical or core laboratories. Numerous published methods require complex sample preparation methods, specialized mass spectrometers and cumbersome or proprietary software tools, thus cannot be easily implemented in unspecialized laboratories. Here, we describe a robust and rapid SS mapping approach that utilizes readily available reagents, instruments, and software; it can be easily implemented in any analytical core laboratories, and tested for its impact on the research community.

Keywords: HCD; Pepsin; Protein disulfide bond; SIM-XL; Tandem mass spectrometry; Trypsin.

Fig. 1.. Comparison of the digestion efficiency between trypsin and pepsin/trypsin.

Non-reduced lysozyme or RNase A was digested with trypsin at pH 6.5, either alone or after a 4-h pepsin digestion at pH 1.3. The resulting peptides were analyzed by LC/MS/MS. The chromatograms were normalized to the same scale. The base peak ion chromatograms of (a) lysozyme or (b) RNase A, digested with either trypsin alone (upper panels) or pepsin/trypsin (lower panels) are shown. Comparing the peptide peaks in the chromatograms of both proteins, pepsin/trypsin produced more peptides than trypsin alone.

Fig. 2.. Comparison of the numbers of SS identified at different tryptic digestion pH with alkylation.

After a 4-h pepsin digestion of lysozyme or RNase A at pH 1.3, the resulting peptides were alkylated with NEM and further digested with trypsin, at various pH conditions as indicated. The peptides were analyzed by LC/MS/MS and the SS-containing peptides were identified by SIM-XL. Different peptides containing identical SS were consolidated into a minimal number SS identified for each protein. The number of total (blue bars) and known (orange bars) SS identified from (a) lysozyme or (b) RNase are scaled to the left Y-axis. The % of the known/total SS (yellow line) identified from (a) lysozyme or (b) RNase A is scaled to the right Y-axis. The most known SS and % of the known/total SS from both proteins were identified at pH 6.5. % Known/total: [(known SS)/(total SS)] X100%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 3.. Using SIM-XL for manual validation of SS.

(a) Through the 2D map, it is possible to assess all identified mass spectra and visualize which protein regions are interacting via SS. (b) An example of a validated MS/MS spectrum of a previously unknown SS (C³⁹²-C⁵³⁷) between two peptides in BSA. Similarly, a manual validation can be done for each identification, considering the fragment peaks matched, the identified residues, and the RANSAC curve [43].

Fig. 4.. Examples of manual validation of SS identification from the HCD spectra.

(a) An MS/MS spectrum of a 3H⁺ ion at m/z 848.08 matched to the lysozyme peptide^22–33 linked to peptide^115–125, with a SS between Cys³⁰ and Cys¹¹⁵, per a SIM-XL score of 2.47. The strings of b- and y-series ions from lysozyme. The mass difference of 1376.63 amu between the y³ and y⁴ fragment ions of 22-GYSLGNWVCAAK-33 provided a strong piece of evidence to validate the SS linkage between these two peptides via Cys³⁰ and Cys¹¹⁵. (b) An MS/MS spectrum of a 3H⁺ ion at m/z 590.59 matched to the lysozyme peptide^6–13 linked to peptide^62–68, with a SS between Cys⁶ and Cys⁶⁴, per a SIM-XL score of 1.32. Since there were not sufficient ions to unambigiously identify either peptide, the SS assignment was not validated. (c) An MS/MS spectrum of a 4H⁺ ion at m/z 623.04 matched to the RNase A peptide^66–75 linked to peptide^113–124, with a SS between Cys⁶⁶ and Cys¹²¹, per a SIM-XL score of 2.46. The strings of b- and y-series ions from the spectrum matched to 66-CKPVNTFVHE-75 and 113-TGSSKYPNCAYK-124 in RNase A, and provided a solid piece of inferential evidence to validate the SS linkage between these two peptides via Cys⁶⁶ and Cys¹²¹. (d) An MS/MS spectrum of a 4H⁺ ion at m/z 791.57 matched to the RNase A peptide^37–57 linked to peptide^118–124, via a SS between Cys⁵² and Cys¹²¹, with a SIM-XL score of 2.02. Since there were many high intensity ions that are unaccounted for, the SS assignment was not validated. The underlined ions derived from the asymmetric SS cleavages were assigned manually.

Fig. 5.. An application of the pepsin/trypsin/SIM-XL method to identify SS in BSA.

(a) Known BSA disulfide bonds identified in this study (red lines). (b) A representative MS/MS spectrum of a BSA peptide⁶⁶⁻⁸⁸, containing an intra-chain SS between Cys⁷⁷-Cys⁸⁶, with a SIM-XL score of 2.49. The strings of b- and y-series ions from the spectrum matched to 66-LVNELTEFAKTCVADESHAGCEK-88, and provided a strong piece of inferential evidence to validate the SS linkage between Cys⁷⁷ and Cys⁸⁶. (c) A representative MS/MS spectrum of BSA peptide^411−420 linked to peptide^456−468 via an inter-chain SS between Cys⁴¹⁵ and Cys⁴⁶¹, with a SIM-XL score of 2.93. The strings of b- and y-series ions from the spectrum matched to 411-IKQNCDQFEK −420 and 456-VGTRCCTKPESER-468, and provided a strong piece of inferential evidence to validate the SS linkage between Cys⁴¹⁵ and Cys⁴⁶¹. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)