intact and native mass analysis of glycoproteins...properdin is a component of the complement system...
Post on 16-Mar-2021
1 Views
Preview:
TRANSCRIPT
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.
top related