The document outlines strategies for bioanalysis of proteins using liquid chromatography-mass spectrometry (LC-MS), highlighting key steps such as sample purification, analyte processing, and the specifics of LC-MS methods. It details various purification techniques, analyte processing steps, and LC-MS fragmentation methods to enhance detection and analysis of proteins and their characteristics. Additionally, it provides examples and considerations for target selection and sample requirements in LC-MS workflows.

1. Strategies for bioanalysis of proteins
using LC-MS
Anne Kleinnijenhuis
anne.kleinnijenhuis@triskelion.nl

2. General workflow
1. Sample purification / pre-processing
2. Analyte processing
3. Liquid Chromatography – Mass Spectrometry (LC-MS)
Parallel:target selection and stable isotope labeled internal
standard (SIL IS) synthesis
Triskelion general workflow for development of protein
LC-MS methods

3. 1. Sample purification / pre-processing
Relatively pure protein solutions
Proceed to analyte processing directly or
proceed to intact protein LC-MS.
No sample purification

4. 1. Sample purification / pre-processing
Protein precipitation
Solid phase extraction
Molecular cut-off filter
SDS-PAGE
Various types of chromatography
Depletion
Less to moderately selective purification

5. 1. Sample purification / pre-processing
Protein A, G (Fc), protein L (κ-LC)
Immobilized receptor
Immobilized antigen
Selective purification
Source: https://www.abdserotec.com/binding-affinities.html

6. 1. Sample purification / pre-processing
Immobilized anti-idiotypic antibody
Not always required in bottom up approach
as LC-MS/MS provides further specificity.
Essential in intact protein (LC-)MS
bioanalysis
Highly selective purification

7. 1. Sample purification / pre-processing
Biotinylation - streptavidin
Poly-His tag – immobilized metal ion e.g. cobalt
Other
Immobilization

8. 1. Sample purification / pre-processing
Magnetic beads – highly flexible
Column format off-line (e.g. micro-tips)
Column format on-line
Purification format

9. 1. Sample purification / pre-processing
Elution => stringent if required but compatible with workflow
Could be part of analyte processing => elution / denaturation
Analyte processing on-bead
Elution

10. 1. Sample purification / pre-processing
Solubilization
Harsh chemical conditions, solvent, pH, reductor
Sonification
High / low temperature
Dispomix
Grinding in liquid nitrogen
Surfactant
Desalting
Addition of protease inhibitors
Other pre-processing

11. 2. Analyte processing
Often the detection target differs from the initial
analyte protein => analyte processing.
Each analyte processing step should be optimal
to obtain a rugged method.
Detection target

12. 2. Analyte processing
None => (intact / native (LC-)MS). Top down.
Deglycosylation => focus signal
Payload cleavage (ADC) => determine conjugated payload /
DAR
Denaturation => make protein accessible to further processing:
heat, chemical, surfactant
Reduction => cleave S-S bridges
Alkylation => modify free SH
Examples

13. 2. Analyte processing
Digestion => several enzymes possible. Trypsin very
compatible with MS, release signature or generic peptide.
Multiple cycles => for resistant / cross-linked proteins
Examples
Name Cleave Does not
cleave
N or C
term
Trypsin KR P C
Chymotrypsin FYWL C
CNBr M C
Lys-C K P C
Glu-C E (and D) P C
Asp-N D N

14. 3. LC-MS
Conventional UPLC (typical ID 2.1 mm)
Robust, fast
Microflow or µLC (typical ID 0.15 mm)
Sensitive
Special sample requirements
Sample composition vs. peptide properties and
chromatographic performance.
NanoLC (typical ID 0.05 mm)
Chromatography

15. 3. LC-MS
Analyzer type
QqQ, Orbitrap, qToF, FT-ICR
Full scan, SIM, MRM
Quantitative / qualitative
Native / intact / bottom up
Multiplex
Mass spectrometry
Buscher et al.
J Res Anal 2015 1 3-10
Infliximab in mouse serum ultrafiltrate.
LC-Orbitrap sum 9 m/z ranges

16. 3. LC-MS
Fragmentation method CID / ETD
CID: intense b, y ions, immonium ions
ETD: less selective cleavage, c, z ions
Peptide mapping (PTMs)
Fragmentation prediction or experimental assessment
MS/MS

17. 3. LC-MS
Fragmentation pHis peptide
Kleinnijenhuis et al.
Anal Chem 2007 79 7450-6

18. 3. LC-MS
Sample purity
Separation of peptide species and peak shape
Ionization efficiency
Targeted / full scan / tandem MS
Fragmentation channels
Chemical noise level
Sensitivity
Linearity / dynamic range
Summary LC-MS/MS considerations

19. Parallel: target selection and SIL IS
Bottom up – intact / native
Peptide requirements
SIL IS peptide vs. SIL IS protein vs. combined
internal standardization (non-labeled protein +
SIL IS peptide)
Preferably 13C / 15N K or R
Target selection

20. Parallel: target selection and SIL IS
Peptide requirements (optional)
Peptide length (6 to ~20-25)
No adjacent cleavage sites
No methionine (M) and cysteine (C)
No asparagine (N) and glutamine (Q)
(Hyper)variable domain for signature peptide
No conjugation sites (PTM, payload)
Many more, study-specific

21. µLC-MS example Kleinnijenhuis et al.
Bioanalysis 2016 8 891-904
Sample requirements
IAA trace UPLC-MS
IAA trace µLC-MS
Column selection CSH / BEH / HSS C18
130 Å, 1.7/1.8 µm, 0.15 x 50 mm

22. Cone flow optimization
µLC-MS example

23. Calibration data
Run Range
(ng/ml)
Points removed
(out of 9)
Intercept Slope
1 5-2000 1 +0.0051 0.00253
2 5-10000 1 -0.0011 0.00271
3 5-2000 1 +0.0037 0.00245
4 5-2000 2 -0.0051 0.00242
5 5-2000 2 -0.0011 0.00235
6 5-2000 2 +0.0083 0.00237
Mean 0.00247
RSD (%) 5.4
µLC-MS example
Six curves in one view

24. Anti-idiotype protocol example
Add diluted streptavidin magnetic beads
Calculate required surface area
Wash beads
Biotinylate anti-idiotypic Ab
Load biotinylated anti-idiotypic Ab
Block unoccupied surface
Wash beads
Load blank, QC, calibration and study samples (10 µl plasma)
Wash beads

25. Anti-idiotype protocol example
Elute analyte protein from beads => payload separate method?
Add SIL IS
Denaturate / surfactant
Reduce
Alkylate
Digestion
Prepare sample for LC-MS
LC-MS/MS analysis

26. Mimic ADC DAR determination example
Deconvolution and manual data inspection
High/lower resolution, data quality, monoisotopic / average m/z

27. Triskelion general experimental set up
Optimize sample purification, analyte processing and LC-
MS (magnetic bead format)
Prepare calibration and QC samples using analyte protein
Include SIL IS for analytical variations after protein elution
Relative vs. absolute recovery (MS/MS settings)
Bottom up protein LC-MS

28. Triskelion general experimental set up
Absolute recovery
𝐶𝐸𝑖𝑠 =
𝑉𝐿 𝑖𝑠
𝑉𝐸 𝑖𝑠
𝐶𝐿𝑖𝑠𝐶𝐸 𝑝𝑒𝑝 =
𝑀𝑊𝑝𝑒𝑝 × 2
𝑀𝑊𝑝𝑟𝑜𝑡
𝑉𝐿 𝑝𝑟𝑜𝑡
𝑉𝐸 𝑝𝑟𝑜𝑡
𝐶𝐿 𝑝𝑟𝑜𝑡
𝑦 =
𝐶𝐸 𝑝𝑒𝑝
𝐶𝐸𝑖𝑠
𝑦 = 𝑎𝑥 + 𝑏
Corrections isotopic
disturbance / molar
𝑎 =
2 × 𝑉𝐿 𝑝𝑟𝑜𝑡 × 𝑀𝑊𝑖𝑠
𝑀𝑊𝑝𝑟𝑜𝑡 × 𝑉𝐿𝑖𝑠 × 𝐶𝐿𝑖𝑠
𝐹1 𝑝𝑒𝑝
𝐹1 𝑖𝑠
DILLTQSPAILSVSPGER
DILLTQSPAILSVSPGE[R_13C6
15N4]
[M+3H]3+
633.0 634.0 635.0 636.0 637.0 638.0m/z
0
50
100
RelativeAbundance
636.024
636.357
633.022632.689
636.690633.356
633.690 637.024
635.691 637.358634.024
𝑥 = 𝐶𝐿 𝑝𝑟𝑜𝑡

29. Triskelion protein MS applications
Monoclonal, bispecific antibodies, multiplex (GLP)
Antibody-drug conjugates (quantification, DAR, intact)
Bottom up and intact
Glycosylation pattern
Collagen & elastin
Milk protein
Food protein authenticity
(Therapeutic) peptide analysis
Food enzyme quantification