Multiplex LC-MS analysis to selectively detect different collagen types in fibrotic tissue

Anne Kleinnijenhuisanne.kleinnijenhuis@triskelion.nl

15 November 2017

Contents

Introduction

Collagen types, method design and development

Results pilot study

Conclusions & Future

Triskelion general workflow

1. Sample purification / pre-processing

2. Analyte processing

3. Liquid Chromatography – Mass Spectrometry (LC-MS)

4. Target selection and internal standardization strategy (parallel phase)

The workflow has been applied in many different protein LC-MS method developments (pharma & food)

1. Sample purification / pre-processing

No sample purification

Less/moderately selective purification:Precipitation, SPE, depletion, SDS-PAGEProtein A/G/L, immobilized receptor

Highly selective purification:Anti-idiotype antibody

SolubilizationHarsh chemical conditions, solvent, pH, reductor, surfactantSonicationTemperature

2. Analyte processing

None => Intact / Top down

Removal of groups, such as sugar / payload

Denaturation => make protein accessible to further processing

Reduction => cleave S-S bridges

Alkylation => modify free SH

Digestion => Release signature or generic peptide

Modify initial analyte to detection target

3. LC-MS

Liquid chromatography several options:Injection conditionsStationary and mobile phasesColumn dimensions

Mass spectrometry several options:Ionization, e.g. ESIAnalyzer, e.g. triple quad, OrbitrapTandem MS

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4. Target selection and internal standardization strategy

Theoretical target selection criteria

Experimental target selection criteria

Stable isotope labeled internal standard (SIL IS) peptide vs. SIL IS protein vs. combined internal standardization (non-labeled protein + SIL IS peptide)

Preferably 13C / 15N as stable isotopes

SIL IS facilitates peak assignment in complex chromatograms

Method development (1)

Aim of the study: development of a method to selectively detect collagen types in lung tissue to support fibrosis research

Methods requiring free protein or protein 3D structure not suitable => Bottom up LC-MS

Challenges:Solubilize crosslinked collagenIdentify selective targets for murine collagen types (1, 3-6)

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Solubilization

Trifluoroacetic acid (TFA) necessary to solubilize crosslinked collagen, however subsequent precipitation.

Other tested conditions: 0.1% Rapigest, heating, sonication, grinding, cutting, Dissociator treatment, ultraturrax, 0.1% Tween-20, and combinations of these conditions.

Crucial step: pretreatment with ammonia. Pellet appearance from white to more transparent.

Method development (2)

Digestion (1)

Initially no LC-MS/MS identifications after digestion with trypsin in 100 mM ABC (slightly basic conditions). Pepsin digestion did provide identifications (acidic conditions).

TFA inhibits trypsin? Modified lysines impair tryptic digestion?

Disadvantage of pepsin: rather non-specific. Trypsin is highly preferred due to its compatibility with LC-MS:

Suitable mass range for MS(/MS) detectionBasic residue in cleaved peptides (K or R): protonation

Method development (3)

Method development (4)

Digestion (2)

Trypsin is even more desired in collagen analysis. K and R occur at regular intervals.

Homo sapiens

Collagen 1α1Helical domain

Method development (5)

Digestion (3)

Trypsin is even more desired in collagen analysis.K and R are conserved amino acids.

Codon group involvement in changes between

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Mus musculus

Collagen 1α1Helical domain

Method development (6)

Digestion (4)

Only after ammonia pre-treatment suitable tryptic digestion was observed

Method development (7)

Target selection

Theoretical target selection using selection criteria and database search. Experimental target selection using qualitative LC-MS Orbitrap.

Color range per target

Method development (8)

Targets were assigned for murine collagens 1α1, 3α1, 4α1, 5α1 and 6α2

Method transfer to triple quadrupole LC-MS/MS

Target structure confirmation using Orbitrap MS/MS data, collagen peptide example:

SIL IS synthesis VGApGPAGARDuck collagen 1α2

Pilot study (1)

Lung lobe wet weight (mg +/- 1 sd)Collagen content (µg)

Hyp assay

Lung fibrosis was induced in 12-week old male C57Bl/6 mice by oropharyngeal administration of bleomycin

Pilot study (2)

Collagen type (ratio analyte / SIL IS)

Collagen type normalized to PBS group

ConclusionsThe developed LC-MS methodology allows the investigation of changes in collagen composition in murine lung.

First step to better defined disease analysis (fibrosis).

Collagen types exhibit different behavior after bleomycin exposure.

FutureDetermine the extracted fraction by analysis of hydroxyproline in supernatant and pellet fractions.

Further method qualification. Obtain pure collagen types?

Other tissues / disease states (e.g. liver / NASH).

Acknowledgements

Frédérique van HolthoonBiomolecular MS group

Reinout Stoop Joline AttemaChrista de Ruiter

Sarah Brockbank

Animal species specific quantification of gelatin