Mass Spectrometry - an essential tool to understand and produce proteins Mark Abbott CEO Peak Proteins Ltd
Talk Outline 1) Brief introduction to Peak Proteins Ltd. 2) How we use mass spectrometry to solve problems and analyse the proteins we make 3) Examples of the use of mass spectrometry
Protein Structures; Art and Science! G6b-B is dimerised around a ligand and bound to Fab fragments – more later
Protein production is easy! • The right construct, cell, culture conditions, purification method, analytical methods and storage conditions appropriate for end use. • What does “right” mean? • There is no one right way. • Every protein is unique and requires handling differently including for different end uses. • How do you know/find the right methods? QC and storage
Peak Proteins Proteins are very individual macromolecules We aim to help you understand them better and make them work for you. • Set up by ex-pharma employees, offer decades of expertise in protein reagent supply and protein structure determination. • Based at Alderley Park near Manchester. • Understand the drug discovery process and the need for high quality bespoke proteins and structural data. • Proven ability to work with many protein classes in small molecule and biologics projects. • Our research-based, innovative approach to solving protein requirements differentiates us from ‘off -the- shelf’ protein suppliers.
Protein mass spectrometry at Peak Proteins • SCIEX X500B mass spectrometer • SCIEX Exion LC • SCIEX OS. BioToolKit and BioPharmaView software • Intact mass and peptide mapping • Seamless transition between methods, C4 and C18 column in one oven so intact and peptide mapping can be queued together
What do we get from intact mass analysis? • Simple rpHPLC , ESI-MS analysis • Is the Intact mass as expected? • Are there post-translational modifications? • Glycosylation, disulphide bonds, phosphorylation, proteolytic processing • Has the product been inadvertently “clipped”? � • Are there unexpected modifications? • Are there expected modifications (biotinylation)?
Peptide mapping - process • Band on SDS-PAGE gel ? • Reduce, alkylate and digest • Trypsin, chymotrypsin (Glu- C) • Separate by rpHPLC • ESI-MSMS • Peptide masses and MSMS sequencing using CID • BioPharmaView software to search data against bespoke sequences • Search data using Mascot against UniProt
What do we get from peptide mapping? • Particularly useful when the intact protein won’t fly on ESI - MS or heterogeneity like N-glycosylation prevents clear identification or if the protein is very impure • Confirmation of identity • Post translational modifications • Mutations • Sequence confirmation • Degradation • Contaminant identification
Examples of how we use mass spectrometry • Identification of products in the manufacture of a therapeutic protein • Trouble shooting a purification • Mapping post translational modifications to enable the engineering of a protein suitable for crystallisation
Manufacture of a therapeutic protein • Cytokine with 2 disulphide bonds • Manufacturing process is; • Express as inclusion bodies in E.coli • Solubilise in urea/reductant A • Refold and form disulphide bonds B C • Further purification D • Need to maximise yield of correct product in initial refold • SDS-PAGE gel shows different conditions Intact mass – 19394 (NR); 19398 (Red.) – 2 disulphides Peptide mapping – 1 disulphide detected in D, work still ongoing as needs GluC • Analyse via intact mass, peptide mapping of disulphide bonds.
Trouble shooting a purification Ladder Load kDa 10 11 12 13 14 15 16 18 19 17 200 140 136 110 87 62 51 A B 40 C 30 22 16 7 Peptide mapping Intact mass A – kinase X 38190 – full length protein; 38189.5 measured B – kinase X 35612 – delete N-t 23 aa; 35611.5 measured C – E.coli CRP (host protein)
Recent client project – G6b-B • Prof. Yotis Senis (University of Birmingham) – group studies regulation of platelets. • Requested the X-ray structure of the extracellular domain of the Megakaryocyte and platelet inhibitory receptor G6b (G6b-B). o In complex with the Fab fragment of a potential therapeutic monoclonal in order to help identify epitope for patent application. o In complex with ligand to visualise & better understand binding/activation mechanism.
Platelet function • Platelets are highly reactive anucleated cell fragments. • Produced by megakaryocytes (MK’s) in bone marrow, spleen & lungs • On vascular injury platelets adhere to exposed vascular extracellular matrix and become activated to form hemostatic plug & seal wound. • Must be tightly regulated to avoid hyper-reactivity and indiscriminate blockage e.g. acute coronary heart disease and stroke. • Inhibition partly due to receptors containing i mmunoreceptor t yrosine- based i nhibition m otifs (ITIM’s) e.g. G6b-B
The inhibitory ITIM receptor G6b-B • G6b-B – an ITIM containing receptor highly G6b-B expressed in MK & platelets. • KO mice have grossly distorted platelet function – macrothrombocytopenia IgV • Binds heparin/heparan containing saccharide ligands • Type I transmembrane protein consists of single IgV-like ECD, a transmembrane domain and cytoplasmic tail with ITIM and SFK ITSM motifs. SH2 SH2 P Shp1 SH2 P • Upon ligand binding central tyrosines of ITIM Shp2 PTP ITIM/ITSM are phosphorylated by Src SH2 P ITSM family kinases to become docking site for inactive phosphatases Shp1 & 2. P • Positions active Shp1/2 to dephosphorylate T active Senis et al. Mol Cell Prot 2007 P key components of ITAM signaling pathway Shp1 & attenuate activation signaling. Shp2
G6b-B ECD expression and purification signal extracellular membrane ITIMS 1 18 142 163 241 C-term N-term Juli Warwicker Several ECD constructs generated Protein Scientist • Extracellular domain is single IgV-like domain of ~13kDa • No published X-ray structure & has < 20% homology with IgV family structures in PDB. • One potential N-linked glycosylation site (Asn32). Derek Ogg CSO/Crystallographer • 4 cysteines, at least one disulphide by homology. • A number of G6b-B ECD constructs were expressed transiently in HEK293 cells. • ECD construct encompassing residues 18-133 expressed well. • Purification by cation exchange and size exclusion from culture medium.
G6b-ECD: Initial purification • Initial SDS-PAGE and LC-MS identified S75 SEC SP-Seph protein consisted of 2 species • Upper band with multiple masses between 14-15kDa indicating N- glycosylation at the predicted site Asn32. • Native G6b-B ECD protein did not crystallise. • Need to remove N-glycosylation • The N-linked sugars could be partly S75 chromatogram cleaved with PNGaseF - but difficult to get removal to go to completion. • Therefore generated Asn32->Asp mutant.
Engineering out the glycosylation S75 • N32->D mutant eliminates upper band. • G6b-ECD now appears as single species on SDS- PAGE & LC-MS. • Intact Mass LC-MS data from a Sciex X500B mass spectrometer gives mass of N32->N G6b-ECD at 13410.2Da. • This is +948Da from the predicted mass & consistent with addition of a single common O-linked tetrasaccharide structure: GalNAc Ser/Thr NeuNAc Gal NeuNAc
Crystallisation of N32->D mutant • No crystals were obtained of N32->D G6b ECD mutant alone or in presence of DP12 (dodecasaccharide heparin fragment) • Crystals of Fab-G6b-DP12 complex were obtained but grew very slowly (2-3 months) and only diffracted to ≤4.0Å at Diamond Light Source • At this resolution could clearly see the Fab and some electron density near the CDRs for putatively bound G6b-B but unable to build model • Improve resolution by also removing the O- glycosylation? • Considered sialidase and O-glycosidase but opted Initial Fab-G6b ECD-DP12 crystals against for cost reasons
O-glycosylation • 13 Ser and 5 Thr residues in G6b-B ECD construct any of which in principle could be O-glycosylated • Bioinformatics with NETOGlyc 4.0 on UniProt identifies 4 residues with a “positive” score • All 4 are found close together in a predicted loop region containing 3 Ser & 2 Thr residues • LC-MSMS peptide mapping via chymotrypsin digest and analysis on Sciex X500B instrument identified a 15aa peptide of this loop with + 948Da mass: 66 80 A SSS G T P T VPPLQPF • Consistent with this loop being the site of O-glycosylation - but which residue?
LC-MSMS peptide mapping via chymotrypsin digest • TOF-MS of digest shows peak at 11.5mins, confirming O-glycosylation with 3+ ion ASSSGTPTVPPLQPF • In source fragmentation to give non- glycosylated 2+ ion and free oligosaccharide • In source fragmentation confirms HexNAc NeuAc Hex NeuAc individual saccharides • Which Ser or Thr? GalNAc Ser/Thr NeuNAc Gal NeuNAc
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