A Powerful Tool for Bioanalysis in Complex Matrices
App Note / Case Study
Last Updated: June 24, 2024
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Published: June 20, 2024
Credit: SCIEX
Quantitation of pharmaceutical drugs is often performed in complex matrices and due to challenging matrix contaminants, highly robust analytical techniques are needed to ensure measurements are accurate and precise.
Long durations of stable analytical performance are often advantageous in high throughput bioanalysis environments. Therefore, longer stable sensitivity and reduced downtime based on cleaning are significant benefits.
This app note highlights a system that can minimize downstream ion path contamination, maintaining high levels of sensitivity and stability over an extended period.
Download this app note to discover how to:
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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.
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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
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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.
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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
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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.
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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
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