strategies for bioanalysis of proteins using lc-ms
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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
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1. Sample purification / pre-processing
Relatively pure protein solutions
Proceed to analyte processing directly or proceed to intact protein LC-MS.
No sample purification
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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
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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
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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
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1. Sample purification / pre-processing
Biotinylation - streptavidin
Poly-His tag – immobilized metal ion e.g. cobalt
Other
Immobilization
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1. Sample purification / pre-processing
Magnetic beads – highly flexible
Column format off-line (e.g. micro-tips)
Column format on-line
Purification format
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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
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1. Sample purification / pre-processing
SolubilizationHarsh chemical conditions, solvent, pH, reductorSonificationHigh / low temperatureDispomixGrinding in liquid nitrogenSurfactant
Desalting
Addition of protease inhibitors
Other pre-processing
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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
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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
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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
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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
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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
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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
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3. LC-MSFragmentation pHis peptide
Kleinnijenhuis et al.Anal Chem 2007 79 7450-6
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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
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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
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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
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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
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Cone flow optimization
µLC-MS example
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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
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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
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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
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Mimic ADC DAR determination exampleDeconvolution and manual data inspection
High/lower resolution, data quality, monoisotopic / average m/z
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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
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Triskelion general experimental set upAbsolute recovery
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Corrections isotopic disturbance / molar
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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