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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
1. Sample purification / pre-processing
Relatively pure protein solutions
Proceed to analyte processing directly or proceed to intact protein LC-MS.
No sample purification
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
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
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
1. Sample purification / pre-processing
Biotinylation - streptavidin
Poly-His tag – immobilized metal ion e.g. cobalt
Other
Immobilization
1. Sample purification / pre-processing
Magnetic beads – highly flexible
Column format off-line (e.g. micro-tips)
Column format on-line
Purification format
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
1. Sample purification / pre-processing
SolubilizationHarsh chemical conditions, solvent, pH, reductorSonificationHigh / low temperatureDispomixGrinding in liquid nitrogenSurfactant
Desalting
Addition of protease inhibitors
Other pre-processing
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
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
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
3. LC-MS
Conventional UPLC (typical ID 2.1 mm)Robust, fast
Microflow or µLC (typical ID 0.15 mm)SensitiveSpecial sample requirements
Sample composition vs. peptide properties and chromatographic performance.
NanoLC (typical ID 0.05 mm)
Chromatography
3. LC-MS
Analyzer typeQqQ, Orbitrap, qToF, FT-ICRFull 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
3. LC-MS
Fragmentation method CID / ETDCID: intense b, y ions, immonium ionsETD: less selective cleavage, c, z ions
Peptide mapping (PTMs)
Fragmentation prediction or experimental assessment
MS/MS
3. LC-MSFragmentation pHis peptide
Kleinnijenhuis et al.Anal Chem 2007 79 7450-6
3. LC-MS
Sample puritySeparation of peptide species and peak shapeIonization efficiencyTargeted / full scan / tandem MSFragmentation channelsChemical noise levelSensitivityLinearity / dynamic range
Summary LC-MS/MS considerations
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
Parallel: target selection and SIL ISPeptide 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
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
Cone flow optimization
µLC-MS example
Calibration dataRun Range
(ng/ml)Points removed
(out of 9)Intercept Slope
1 5-2000 1 +0.0051 0.002532 5-10000 1 -0.0011 0.002713 5-2000 1 +0.0037 0.002454 5-2000 2 -0.0051 0.002425 5-2000 2 -0.0011 0.002356 5-2000 2 +0.0083 0.00237
Mean 0.00247RSD (%) 5.4
µLC-MS example
Six curves in one view
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
Anti-idiotype protocol exampleElute analyte protein from beads => payload separate method?
Add SIL IS
Denaturate / surfactant
Reduce
Alkylate
Digestion
Prepare sample for LC-MS
LC-MS/MS analysis
Mimic ADC DAR determination exampleDeconvolution and manual data inspection
High/lower resolution, data quality, monoisotopic / average m/z
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
Triskelion general experimental set upAbsolute recovery
𝐶𝐸𝑝𝑒𝑝=(𝑀𝑊 𝑝𝑒𝑝×2𝑀𝑊 𝑝𝑟𝑜𝑡 )( 𝑉𝐿𝑝𝑟𝑜𝑡
𝑉𝐸𝑝𝑟𝑜𝑡 )𝐶𝐿𝑝𝑟𝑜𝑡
𝑦=(𝐶𝐸𝑝𝑒𝑝
𝐶 𝐸𝑖𝑠)
Corrections isotopic disturbance / molar
DILLTQSPAILSVSPGER
DILLTQSPAILSVSPGE[R_13C615N4]
[M+3H]3+
633.0 634.0 635.0 636.0 637.0 638.0m/z0
50
100
Rela
tive
Abun
danc
e
636.024636.357
633.022632.689
636.690633.356
633.690 637.024635.691 637.358634.024
𝑥=𝐶𝐿𝑝𝑟𝑜𝑡
Triskelion protein MS applicationsMonoclonal, bispecific antibodies, multiplex (GLP)Antibody-drug conjugates (quantification, DAR, intact)Bottom up and intactGlycosylation patternCollagen & elastin Milk proteinFood protein authenticity(Therapeutic) peptide analysisFood enzyme quantification
