A Powerful Tool for Bioanalysis in Complex Matrices

p 1 For research use only. Not for use in diagnostics procedures. - Improving complex phosphopeptide characterization with hybrid EAD/CID MS/MS fragmentation Jeremy Potriquet1 , Patrick Pribil2 and Daniel Winter3 1SCIEX Australia; 2SCIEX Canada; 3All G Foods This technical note describes the use of combined electron activated dissociation (EAD) and collision-induced dissociation (CID) on the ZenoTOF 7600 system to enhance the fragmentation of peptides from casein tryptic digests (Figure 1). In many organisms, proteins can exist in multiple isoforms and have diverse post-translational modifications (PTMs). Characterizing proteins invariably requires a deep understanding of the nature of these PTMs and their effects on protein structure and biological function. Mass spectrometry can be used for PTM characterization through different modes of fragmentation of modified peptides. Compared with EAD or CID alone, using a hybrid EAD/CID fragmentation approach improved the sequence coverage for casein peptides and the characterization of PTMs on these peptides. In particular, the hybrid EAD/CID approach was useful in differentiating between multi-phosphorylated peptide isomers, leading to the unambiguous assignment of phosphorylation sites in casein peptide sequences. We demonstrate that this differentiation can be achieved at high acquisition speeds, with limited sample preparation and without the need for derivatization. Key features of the ZenoTOF 7600 system for phosphopeptide characterization • Zeno trap pulsing allows accumulation of ions during TOF pulsing for enhanced duty cycle, generating higher quality MS/MS spectra for low abundant targets such as phosphopeptides • EAD fragmentation preserves phosphorylation sites and accurate positional information due to the generation of c and z+1 fragment ions • When EAD is combined with CID, nearly full sequence coverage can be achieved even with larger peptides with multiple modifications • PTM data processing can be performed using SCIEX OS software and is compatible with other processing softwares such as Skyline software, MSFragger algorithm and Peaks Studio software Figure 1. Concept of hybrid EAD/CID fragmentation on the ZenoTOF 7600 system. p 2 For research use only. Not for use in diagnostics procedures. Introduction Caseins are an important component of milk and play a major role in diets worldwide, as they are highly nutritious and provide essential amino acids. Milk proteins include 4 caseins (αs1- casein, αs2-casein, β-casein and κ-casein) and 2 major whey proteins (α-lactalbumin and β-lactoglobulin). 1 Casein studies have shown that this group of proteins can have extremely complex PTMs with αs1-casein harboring 9-10 phosphorylations, αs2-casein harboring 10-13 phosphorylations and β-casein harboring 5 phosphorylations. In addition, glycosylations and many genetic variants in which specific amino acid replacements occur can change protein functionality.2 Studies have shown that phosphorylation of the casein proteins plays a critical role in the formation of casein micelles and in the interaction with, and transport of, divalent cations such as calcium and zinc. These casein functions are important to facilitate the adsorption of these nutrients in the gut of the nursing infant.3,4,5,6 Characterizing phosphorylation sites on casein proteins is inherently difficult because casein-containing mixtures include peptides with highly variable levels of phosphorylation. Overcoming the resulting ionization suppression in positive-ion mass spectrometry (MS) often requires the derivatization of phosphoserines. 7 These analysis limitations are significant because recent improvements in recombinant DNA technology allow the possibility for large-scale production of recombinant milk proteins. With the significant economic interest and growing need for alternative sustainable food production, it is essential to accurately and rapidly monitor milk batches for genetic variants and to characterize the sites and relative ratios of phosphorylations. Here, the ability to perform a hybrid EAD/CID fragmentation is demonstrated to provide enhanced sequence coverage and greater confidence in assigning phosphorylation sites on casein proteins with complex phosphorylation profiles. Methods Sample preparation: Casein proteins (native β-casein, αS1- casein and αS2-casein) were purified from cow’s milk by precipitation under low-pH conditions, then solubilized in 6 M urea and further purified by ion exchange. Samples were digested with trypsin, desalted in pure water, lyophilized and resuspended in water with 0.1% formic acid to the desired concentration. Sample loadings ranged from 400 ng to 700 ng. Chromatography: Separations were performed using a Waters ACQUITY UPLC M-Class system plumbed for microflow chromatography (7 µL/min) and operated in direct-inject mode. The analytical column was a ProteCol PEEKSIL C18G column (3 µm, 200 Å, 250 x 0.3 mm). Column temperature was controlled at 40°C. A 25-min gradient was used for all data-dependent acquisition (DDA) experiments, as shown in Table 1. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. Mass spectrometry: Data were acquired using the ZenoTOF 7600 system in DDA mode. TOF MS scans were 200 ms across a mass range of 400-1750 m/z. The MS/MS scan range was 100-2000 m/z using a hybrid EAD/CID fragmentation approach with 65 ms accumulation times and 30 ms reaction times. An electron beam current setting of 3000 nA and an electron KE setting of 2 eV were used. Dynamic collision energy (CE) was applied for CID and hybrid EAD/CID. Source conditions included 20 psi for GS1, 15 psi for GS2, 35 psi for curtain gas, 5000 V for spray voltage and 150°C for source temperature. The top 15 candidates were selected for MS/MS with charge states from 2 to 4, with an exclusion of 5 s after 1 occurrence. Data processing: Data were processed with Skyline software using the DDA peptide search tool using the MSAmanda search engine with 25 ppm MS1 and MS2 mass tolerances and the MSFragger algorithm. Spectrum identifications were manually confirmed and annotated using SCIEX OS software. Combining EAD and CID fragmentation to generate information-rich spectra The ZenoTOF 7600 system allows either EAD fragmentation alone or the ability to do consecutive EAD and CID fragmentation on isolated precursors, with no difference in overall cycle times between these 2 approaches. Hybrid EAD/CID fragmentation generates richer MS/MS spectra which can increase confidence in sequence assignments, as shown in Table 1. Chromatographic gradient for peptide separations. Time (min) Mobile phase A (%) Mobile phase B (%) 1.0 97 3 20.0 97 3 20.1 20 80 22.0 20 80 22.1 97 3 25.0 97 3 p 3 For research use only. Not for use in diagnostics procedures. Figure 2 for the example β-casein peptide FQSEEQQQTEDELQDK. While EAD fragmentation provided excellent sequence coverage with MS/MS spectra that have predominantly c, z, z+1 and z+2 fragments, using hybrid EAD/CID fragmentation generated more diverse ions, especially in the low-mass region. Increased sequence coverage using hybrid EAD/CID for challenging larger peptides Combining EAD and CID fragmentation is also valuable for increasing sequence coverage on larger peptides, which might be harder to fragment (Figure 3). The αS1-casein peptide QFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEK contains 42 amino acids and 4 proline residues and benefits significantly from the use of hybrid EAD/CID fragmentation. The observed abundance of b, c, y, z+1 and z+2 fragment ions provides near complete sequence coverage, including for the regions of the peptide with proline residues that are typically challenging to sequence with EAD fragmentation. Phosphorylation site differentiation and multi-phosphorylated peptide sequence coverage When analyzing mixtures of isomeric phosphopeptides, chromatographic separation is a key method used to overcome ionization suppression of some of the phosphorylated variants. As shown in Figure 4, the 3 isomeric variants of the triplephosphorylated αS2-casein peptide NTMEHVSSSEESIISQETYK were chromatographically resolved through separation using a relatively long gradient on a 250 mm analytical column. Using a DDA approach with hybrid EAD/CID fragmentation allowed the identification of all the different positional variants using the MSFragger algorithm. It was subsequently confirmed with Skyline software using the MSAmanda search function against a FASTA casein database. The results were annotated using SCIEX OS software as shown in Figure 4. Notably, excellent complementarity of fragment ion information was observed between b, c, y, z, z+1 and z+2 ions, with the c, z+1 and z+2 ion series contributing most significantly to the correct assignment of the modifications. No loss of fragment ions carrying labile modifications was observed when combining EAD and CID fragmentation. Figure 2. Effect of hybrid EAD/CID fragmentation on the MS/MS spectra for the β-casein peptide FQSEEQQQTEDELQDK. The theoretical fragment ions for this peptide are shown in the table. The fragment ions observed in the MS/MS spectra that matched the theoretical fragment ions are highlighted in red. p 4 For research use only. Not for use in diagnostics procedures. Figure 3. Comparison of MS/MS spectra and sequence coverage between the CID and hybrid EAD/CID fragmentation for the αS1-casein peptide QFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEK. The theoretical fragment ions for this peptide are shown in the table. The fragment ions observed in the MS/MS spectra that matched the theoretical fragment ions are highlighted in red. CID Hybrid EAD/CID p 5 For research use only. Not for use in diagnostics procedures. The same observation was made when targeting the multiphosphorylated αS1-casein peptide QMEAESISSSEEIVPNSVEQK, which consisted of 21 amino acids, 5 phosphorylation sites and a proline residue. Figure 5 shows that 100% sequence coverage was achieved with this challenging peptide and all the correct modification sites were confirmed through the resulting c and z+1 fragment ion evidence. Conclusions • EAD or hybrid EAD/CID fragmentation on the ZenoTOF 7600 system can be used to elucidate complex peptide sequences and correctly assign PTMs, such as phosphorylation • A single DDA experiment with a 25-minute gradient was sufficient to acquire all the information necessary to identify and differentiate isobaric phosphorylation permutations on peptide sequences from a casein tryptic digest • Combining CID and EAD fragmentation allows for the generation of information-rich MS/MS spectra with excellent sequence coverage while retaining PTM positional information • The hybrid EAD/CID method can easily be converted into a targeted multiple reaction monitoring (MRMHR) method on the ZenoTOF 7600 system to quantify and rapidly monitor the different phosphopeptide variants in casein extracts for dayto-day quality controls • To date, the complete mapping of phosphorylation from casein samples has not been achieved without peptide enrichment. In this experiment, the detection and characterization of the different expected isoforms of phosphorylated caseins was possible, which allows for future development of MS-based quantitation methods for such peptides Figure 4. Extracted ion chromatogram (XIC) and MS/MS sequence coverage with phosphorylation site differentiation for the multiphosphorylated αS2-casein peptide NTMEHVSSSEESIISQETYK. The green boxes denote the variable phosphorylated region (3 successive serine residues, of which 2 were phosphorylated for a given peptide), highlighting the distinctive c ions for a given peptide. Figure 5. Hybrid EAD/CID MS/MS spectrum and sequence coverage for the multi-phosphorylated αS1-casein peptide QMEAES[Pho]IS[Pho]S[Pho]S[Pho]EEIVPNS[Pho]VEQK. p 6 For research use only. Not for use in diagnostics procedures. References 1. Walstra, P., Walstra, P., Wouters, J.T.M., Geurts, T.J. (2005). Dairy Science and Technology (2nd ed.). CRC Press. https://doi.org/10.1201/9781420028010 2. Bijl E, Holland JW, Boland M. (2020). Posttranslational modifications of caseins (3rd ed.) Elsevier Press. https://doi.org/10.1016/B978-0-12-815251-5.00005-0 3. Dingess, K.A., Gazi I., van den Toorn H.W.P., Mank M., Stahl B., Reiding K.R., Heck A.J.R. (2021). Monitoring Human Milk β-Casein Phosphorylation and O-Glycosylation Over Lactation Reveals Distinct Differences between the Proteome and Endogenous Peptidome. Int. J. Mol. Sci. 2023;22:8140 4. Molinari C.E., Casadio Y.S., Hartmann B.T., Arthur P.G., Hartmann P.E. (2013). Longitudinal analysis of protein glycosylation and beta-casein phosphorylation in term and preterm human milk during the first 2 months of lactation. Br. J. Nutr.2013;110:105-115 5. Nadugala, B.H., Pagel, C.N., Raynes, J.K., Ranadheera, C.S., Logan, A. (2022). The effect of casein genetic variants, glycosylation and phosphorylation on bovine milk protein structure, technological properties, nutrition and product manufacture. Int. Dairy J. 2022;133:105440 The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use. Rx Only. Product(s) not available in all countries. For information on availability, please contact your local sales representative or refer to https://sciex.com/diagnostics. All other products are For Research Use Only. Not for use in Diagnostic Procedures. Trademarks and/or registered trademarks mentioned herein, including associated logos, are the property of AB Sciex Pte. Ltd. or their respective owners in the United States and/or certain other countries (see www.sciex.com/trademarks). © 2023 DH Tech. Dev. Pte. Ltd. MKT-29611-A