In this presentation important aspects of target selection and internal standardization in protein LC-MS are discussed. In addition there are 3 slides about coronavirus protein LC-MS considerations.

1. Target selection and internal standardization
Episode 7
Frédérique van Holthoon
March 2020 Anne Kleinnijenhuis

2. Introduction
●Protein LC-MS often involves analyte processing in order to:
●Make the analyte detectable e.g. proteins in networks
●Improve the quantitative analytical performance
●Remove or avoid heterogeneity of analyte
●Examples of analyte processing steps
●Physical: heating, sonication
●Chemical: reagents, pH
●Enzyme treatment
●Target selection using high resolution non-targeted LC-MS

3. Target types
●Quantitative targets
●Unique or signature targets
●Generic targets
●Qualitative targets
●Analogous targets
●Subtargets
●Multiple targets

4. Selection criteria
●Target selection criteria are typically related to:
●LC-MS performance
●Target unambiguity
●Target stability
●Target uniqueness
●Chemical and enzymatic reaction efficiencies
●Required target coverage related to (potential) source
materials

5. Unique targets
●To selectively quantify proteins
●Targets are unique considering:
●Other analyte proteins
●Other components in the tissue of interest
●Optional: other tissues and/or other animal species
●Often originate from a variable region
●SIL IS required for quantitative method

6. Generic targets
●To collectively quantify proteins
●Target occurs in a group of analytes
●Consider the evolutionary divergence
●Often originate from a conserved region
●SIL IS required for quantitative method
●Unique targets can still be used in parallel to individually quantify
protein group members

7. Analogous targets
●When a SIL IS is not available for all targets of interest, analogous
targets, originating from exactly the same positions in the aligned
sequences, may be utilized
●Consider the evolutionary divergence
●Select 1 quantitative target + SIL IS
●Suitable for qualitative monitoring of:
●Orthologs (separated by speciation event)
●Paralogs (separated by duplication event)

8. Subtargets
●Only a generic quantitative target shows good performance
●Uniqueness of the generic target is not sufficient
●Subtargets are unique but have worse performance, e.g. regarding
sensitivity or chromatographic peak shape
●Subtargets are analyzed qualitatively to monitor the presence of
individual analyte proteins
●Example: collagen from closely related species, such as Bos taurus
and Bubalus bubalis

9. Utilization of multiple targets (1)
●The possibilities of LC-MS regarding targets are virtually unlimited
●A single method might contain several of the target types
2.5 3 3.5 4 4.5 5 5.5 6 6.5
Retention time (min)
RAT SUS MACFA
MUS BOV RABIT
CANLF FELCA HUM
Example: monitoring of blood protein targets from several animal species

10. Utilization of
multiple targets (2)
●Targets can be used for any purpose, also to detect different
parameters and even non-biochemicals
●Determine protein integrity (after selective purification)
●Assess the sample purification performance e.g. monitor generic
IgG target after protein A and/or G
●Qualitative targets can be upgraded to quantitative targets by SIL IS
incorporation and absolute recovery assessment

11. Quantitative LC-MS bioanalysis
●After target selection transfer to triple quadrupole LC-MS/MS
●Triple quadrupole has good performance regarding sensitivity,
precision and dynamic range, which are essential for bioanalytical
methods used in (pre-)clinical studies
●Considerations for quantitative LC-MS bioanalysis:
●Guidelines for method validation
●Dynamic range
●Chemical noise
●Accountability / SIL IS
●Relative and absolute recovery

12. Method validation
●The required set up and criteria of either chromatographic or ligand
binding assays (LBA) are a basis for the validation
●For protein LC-MS, which often includes several chemical and
enzymatic purification and/or processing steps, it is acceptable to use
LBA criteria (20/25%)
●Validate other parameters relevant to the particular assay when
they are not described in the guidelines (scientific validation)

13. Dynamic range
●Triple quadrupole: > 6 orders
of magnitude dynamic range in
solvent, in samples containing
(remnants of) biological matrix
often 3-4 orders of magnitude
●Large dynamic range = less reanalyses = green, more efficient &
cost effective
●Include other targets, quantifiers and/or qualifiers with varying
sensitivity to expand dynamic range or to be able to change when
there are issues

14. Chemical noise
●Especially in biological matrices there can be varying background
chemical noise, affecting the dynamic range and specificity
●Include other targets, quantifiers and/or qualifiers to be able to
change when there are background issues
●SIL IS is essential to match analyte peak, when there is
background from the biological matrix, retention time shift and/or
when highly similar protein targets are analyzed

15. Accountability (1)

16. Accountability (2)
●Peptide A and B originate from the same
analyte protein
●Stable isotope labeled (SIL) peptide A and B were added as mix
●The sample was analyzed in duplicate. Missing (SIL) Peptide B
signal in one of the replicates. Possibly explained by temporarily
malfunctioning electrospray
●This example demonstrates that SIL IS should be added for each
target of interest in order to obtain fully accountable results in LC-MS

17. Relative and absolute recovery
●Relative recovery is calculated against matrix-matched calibration
curve prepared with analyte protein
●Absolute recovery is calculated using the theoretical signal ratio
(analyte / SIL IS) at 100% recovery
●Preferably the absolute recovery is close to 100%. It can be
acceptable to only have relative recovery close to 100%
𝑎 =
𝑛 × 𝑉𝐿 𝑝𝑟𝑜𝑡 × 𝑀𝑊 𝐼𝑆
𝑀𝑊𝑝 𝑟𝑜𝑡 × 𝑉𝐿 𝐼𝑆 × 𝐶𝐿 𝐼𝑆
𝐹1 𝑝𝑒𝑝
𝐹1 𝐼𝑆
a
n
VLprot
MWIS
MWprot
VLIS
CLIS
F1pep
F1IS
Slope at 100% absolute recovery
Peptide copy number
Load volume protein
Molecular weight internal standard
Molecular weight protein
Load volume internal standard
Load concentration internal standard
Fraction first isotope peptide
Fraction first isotope internal standard

18. Concluding remarks of part 1
●Several considerations for target selection and internal
standardization in protein LC-MS were presented
●Target selection is an important procedure to obtain a suitable and
informative method
●Internal standardization is essential to obtain acceptable and
accountable results on the single sample level
Next slides: part 2
about coronavirus
protein LC-MS
considerations

19. Roadmap to SARS-CoV-2 spike protein quantification (1)
●Purify protein/virus from biological matrix using immobilized antibody
(optional) and appropriate safety measures
●Theoretical target selection criteria (strict): part of the chain, between 6
and 18 amino acids (sensitivity), no M (partial oxidation), no C
(carbamidomethylation), no N (possible glycosylation, deamidation), no
terminus, no adjacent cleavage sites, no Q (deamidation), not too many
acidic residues (easier protonation)
●Multiple candidates identified (from entry NC_045512.2): GVYYPDK,
GWIFGTTLDSK, SFTVEK, DIADTTDAVR and VTLADAGFIK
●Corresponding peptides in SARS-CoV-1 (entry P59594): GVYYPDEIFR,
GWVFGSTMNNK, SFEIDK, DVSDFTDSVR and VTLADAGFMK

20. ●Consider evolutionary divergence by comparison to other strains, bat
coronavirus, SARS-CoV-1 et cetera and investigate potential mutation routes
●Denature, reduce, alkylate, digest and prepare for LC-MS using appropriate
safety measures
●Optimize every method module or use optimized modules. Drop selection
criteria (e.g. no Q) when too many candidate targets are not suitable
●Investigate required target uniqueness in relation to goal of the study,
source tissue and selectivity of the purification
●Synthesize SIL IS and assess absolute recovery prior to validation
Roadmap to SARS-CoV-2 spike protein quantification (2)

21. ●Codon table showing the codon usage (blue, expressed as DNA) and
amino acid usage (red) in SARS-CoV-2 spike protein (NC_045512.2), an
illustrative tool to aid in the investigation and comparative monitoring of
(potential) mutation routes
Roadmap to SARS-CoV-2 spike protein quantification (3)

22. SlideShare series Anne Kleinnijenhuis
● 7. Target selection and internal standardization (Mar 2020)
● 6. Integrated hemolysis monitoring for bottom up protein bioanalysis (Aug 2019)
● 5. Animal species specific quantification of gelatin with TrustGel (Nov 2017)
● 4. Domain-specific analysis of collagen code (May 2017)
● 3. Exploring LC-MS peptide dynamic range (Dec 2016)
● 2. Strategies for bioanalysis of proteins using LC-MS (May 2016)
● 1. Proposal for the absolute quantification of modular molecules (Oct 2015)