Strategies for bioanalysis of proteins using LC-MS

Anne Kleinnijenhuisanne.kleinnijenhuis@triskelion.nl

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

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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