Intact and Native Mass Analysis of GlycoproteinsMarshall Bern1 Yong J. Kil1 Tomislav Caval2 Vojtech Franc2 Albert J.R. Heck2

1Protein Metrics Inc. 2Biomolecular Mass Spectrometry and Proteomics, Utrecht University

Contact: bern@proteinmetrics.com

Byologic® FeaturesIntroduction

www.proteinmetrics.com

Byologic® FeaturesResults: EPO

Byologic® FeaturesMethods – Experimental

Methods – Computational

Byologic® FeaturesConclusions

Intact mass analysis provides a simultaneous and quantitative view of a

protein’s major proteoforms, including variations in glycosylation. Intact mass

spectra vary with solution conditions:

• Denaturing conditions give fewer adducts, higher charge states, and

stronger overall signal.

• Native conditions preserve noncovalent binding and original folded

structure of the proteins, which results in fewer and lower charge states.

Lower charge state provides more space between adjacent charge states,

allowing separation of protein ions coming from a heterogeneous mass

distribution.

After data acquisition, m/z spectra are deconvolved to neutral mass spectra.

This computational step is prone to errors and artifacts, especially for the

complex signals of glycoproteins. For the past 25 years, almost all charge

deconvolution has been done with the MaxEnt algorithm (Ferrige et al, 1992),

which is offered in various implementations by MS instrument vendors.

Here we demonstrate the utility of native MS and a new “parsimonious”

charge deconvolution algorithm for the analysis of complex glycoproteins.

Byologic® FeaturesProperdin / Factor P

Byologic® FeaturesCetuximab

Byologic® FeaturesReferences and Acknowledgments

Protein Metrics Intact

mass software can

deconvolve wide m/z and

mass ranges with minimal

artifacts. Colored dots

connect neutral masses to

m/z peaks and let the user

validate masses visually.

Assignments may be

made manually or auto-

matically from calculated

masses or mass deltas.

IdeS and DTT digested

Cetuximab shows LC at ~23 kDa,

Fc/2 at ~25 kDa, Fd at ~27 kDa,

and Fc at ~51 kDa.

We analyzed the following samples:

• Recombinant human erythropoietin EPO BRP

• Purified human Properdin / Factor P

• Cetuximab (trade name: Erbitux)

We chose these targets for their complex glycosylation.

Cetuximab is, to our knowledge, the only therapeutic

mAb currently on the market with Fab glycosylation.

Native MS was performed by direct infusion on an

Exactive Plus Orbitrap instrument with extended mass

range (EMR) (Thermo Fisher Scientific) using a

standard m/z range of 500-10,000. LC-MS/MS was

performed on an Orbitrap Fusion with EThcD

fragmentation. More details are described in a

previous publication (Yang et al, 2016).

Like other charge deconvolution programs (MaxEnt, Bayesian, UniDec),

Protein Metrics Intact starts with an initial guess of the charges in the m/z

spectrum, computes an initial m spectrum along with charge histogram for

each mass, and then iteratively improves the m spectrum and charge

histogram until together they produce a computed m/z spectrum close to the

observed one. MaxEnt aims for a high-entropy m spectrum to improve

resolution, but can also produce artifacts. The new algorithm splits the

problem in two: charge inference, which aims for a minimal or parsimonious

set of masses, and Richardson-Lucy peak sharpening for resolution.

Thermo Exactive Plus EMR was

used for native MS analysis

Color indicates neutral monosaccharide

composition; number gives # NeuAc’s

Charge states slightly overlap

in native MS spectrum of EPO.

Too tall Too small

Previous work (Yang et al, 2016) gave a comprehensive analysis of rhEPO

glycosylation with native mass spectrometry and glycopeptide profiling using

trypsin and GluC digests. Complex native mass spectra (black borders) have

mildly overlapping charge states. Charge deconvolution was performed by

Bayesian Protein Reconstruct tool from BioAnalyst (Sciex) (orange). Here we

reanalyze the data with Protein Metrics Intact (blue).

Overall agreement between Sciex

Bayesian deconvolution and PMI

Intact is good, but PMI Intact’s peak

intensities agree more closely with

the major charge state in the m/z

spectrum, as shown at lower right.

Correctness of intensities of masses

below 28 kDa is hard to judge due to

overlap around m/z 3100.

PMI Intact Deconvolution

Sciex Deconvolution

PMI Intact

Sciex28,326 too small?

Too tall?

Unfolded

Native monomer

Dimer

Properdin is a component of the Complement system with thrombospondin

repeats, C-mannosylation, O-fucosylation, and N-glycosylation. Previous work

(Yang et al, 2016) gave an analysis of properdin relying on an m/z spectrum

rather than a neutral mass spectrum due to the difficulty of deconvolution.

Zoom of wide-

range Thermo

deconvolution

Zoom of wide-

range PMI

deconvolution

Monomer

z = 14+

Narrow mass range

with salt adducts

No adducts

Deconvolution with wide m/z

and mass ranges (right)

surveys the proteoforms.

Thermo Deconvolution

(lower left) gives incorrect

peak intensities and widths.

Deconvolution with a narrow

m/z and mass range (lower

right) gives assignable

monomer masses.

Interestingly, tri-antennary

N-glycans are observed only

on proteoforms with 15 C-

mannosylations.

Intact mass analysis is routinely used to check primary sequence, heavy /

light chain pairing, and glycosylation in both intact and reduced mAbs.

Zoom of 26 – 28 kDa Fd

Exactive EMR spectrum

Zoom of 23 – 28 kDa mass range

Fc with G0F

G1F

+ C-terminal Lys

LC

Fd

Zooms of the neutral mass spectrum show ordinary mAb glycosylation on the Fc, but

more complex N-glycans on the heavy chain variable region (Fd), including Gal-α-Gal,

antennal fucosylation, and tri-antennary glycans.

The Fd mass spectrum is in close

agreement with previous work (Janin-

Bussat et al). Notice, however, that the

Orbitrap spectrum resolves the 27,525 Da

mass, and the peaks at 27,688 and

27,831 are mislabeled (100 Da off) in

Janin-Bussat, missing the antennal Fuc.

QTOF mass spectrum

from Janin-Bussat et al.

For the past 25 years, charge deconvolution has been performed almost

exclusively by some version of MaxEnt. High-resolution native MS and the

need to analyze more complex molecules motivated the development of a

new deconvolution algorithm with the following advantages:

• Wider applicability (any MS instrument, any molecule type)

• Fewer and smaller algorithmic artifacts

• Optional peak sharpening for greater fidelity to raw data

Although the samples were chosen primarily for technology development, the

studies revealed some novel characteristics of properdin and Cetuximab.

• Properdin includes tri-antennary glycans, seemingly only on proteoforms

with 15 C-mannosylations, evidence of correlated PTMs.

• Cetuximab Fab glycosylation includes antennal fucose

Ferrige, A.G.; Seddon, M.J.; Green, B.N.; Jarvis, S.A.; Skilling, J.; Staunton, J.

Disentangling electrospray spectra with maximum entropy, Rapid Comm Mass Spec, 1992.

Yang, Y.; Liu, F.; Franc, V.; Halim, L. A.; Schellekens, H.; Heck, A. J. R. Hybrid mass

spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity.

Nat. Commun, 2016.

Janin-Bussat, M.-C., et al. Cetuximab Fab and Fc N-Glycan Fast Characterization Using

IdeS Digestion and Liquid Chromatography Coupled to Electrospray Ionization Mass

Spectrometry. In Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols;

Beck, A., Ed.; Humana Press: Totowa, NJ, 2013; pp 93–113.

A.J.R.H acknowledges support from the Netherlands Organization for Scientific Research

(NWO) funding the large-scale proteomics facility Proteins@Work. We thank Genmab

(Utrecht) for providing us with mAbs including Cetuximab and Daclizumab.