Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry
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http://hdl.handle.net/2047/D20259955
Agar, Jeffrey (Advisor)
Ivanov, Alexander R. (Committee member)
Zhou, Zhaohui (Committee member)
However, the complexity of protein samples, especially for top-down proteomic applications, necessitates a high resolution separation component prior to characterization by ESI-MS, preferably in an online format. Conventional techniques include LC and ion mobility spectrometry. Reversed phase LC in particular, which separates analytes according to differences in hydrophobicity, is perhaps the most widely-used mode of LC for the separation of proteins online with ESI-MS. An alternative approach, however, involves capillary zone electrophoresis (CZE). CZE enables analyte separation according to charge and hydrodynamic volume differences in the liquid phase. Essentially, CZE may be viewed as a complementary technique that separates analytes according to a different mechanism than LC. Advancements in CZE instrumentation, capillary coatings, and interfaces with ESI-MS that have occurred over the past several decades have enabled the application of this technique for top-down proteomics.
In Chapter 2, a relatively small protein pharmaceutical possessing a single glycosylation site - recombinant human interferon-β1 (rhINF-β1) (Avonex, Biogen)was analyzed under denaturing conditions by top-down CZE-ESI-MS. A positively-charged, cross-linked polyethylenimine (cPEI) coating was administered and used on a commercial sheathless system (CESI, SCIEX) coupled to the Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). The analyses resulted in the identification of 138 individual proteoforms of rhINF-β1, of which 55 were quantitated. Charge species due to sialylation and deamidation were separated by CZE. Moreover, isobaric species due to positional isomers of a sialic acid on biantennary glycans were resolved by CZE. Fragmentation by high energy collision-induced dissociation (HCD) and electron transfer dissociation (ETD) contributed to the identification of many proteoforms. Reduced trapping pressure in the HCD cell of the Orbitrap Elite mass spectrometer enabled for accurate, highly sensitive detection of intact proteins. Quantitative intact analysis of rhINF-β1 revealed a potential correlation between methionine-truncation and deamidation, as well as between deamidation and glycosylation structures. This chapter is based on a 2016 publication in Analytical Chemistry.
In Chapter 3, analysis by sheathless CZE-ESI-MS was extended towards the characterization of a therapeutic monoclonal antibody (mAb). The cPEI coating, however, did not result in sufficient resolution of proteoforms on the mAb either in an intact form or after limited digestion (middle-down). Consequently, an alternative positively-charged coating (1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I)), enabling a lower reverse electroosmotic flow (EOF) for higher resolution separations, was used. In middle-down analyses, charge-based proteoforms of the mAb were separated due to deamidation. Partial separation was achieved between species differing in glycosylation as a result of hydrodynamic volume differences. Tandem mass spectrometry with HCD fragmentation was used to confirm identities of many of the proteoforms. Analysis of the intact molecule revealed mAb populations differing in the extent of glycosylation, with two, one, or zero glycosylation sites per mAb. These populations were resolved baseline by CZE as a result of differences in hydrodynamic volume. Moreover, trace quantities of dissociated free chains of the mAb were detected and resolved by CZE. Quantitation of various populations of the intact mAb was achieved and corroborated between several experimental replicates. These results were further validated in analysis of the mAb performed under native conditions on a polyacrylamide-coated capillary coupled to the Q Exactive Plus MS via the CESI interface.
In Chapter 4, the sheathless CZE-ESI-MS platform was used for the analysis of numerous proteins under native conditions in one of the first demonstrations of CZE coupled online to native MS. Bare-fused silica (BFS) and polyacrylamide-coated capillaries, available commercially from SCIEX, were coupled to Orbitrap mass spectrometers in this application. First, a simple mixture of 3 proteins and associated homodimeric/homotetrameric protein complexes was analyzed under native conditions on a BFS capillary coupled to the Exactive EMR mass spectrometer (Thermo Fisher Scientific). The use of a near-physiological buffer allowed for the mitigation of non-specific adsorption of proteins to the capillary surface. In several instances, protein complexes were partially-resolved from their corresponding monomers by CZE. The use of all-ion-fragmentation allowed for the characterization of mixture components, including prominent proteoforms for each protein/protein complex. A more complex mixture of over 11 proteins was then analyzed, this time on a polyacrylamide-coated capillary coupled to the Exactive EMR mass spectrometer. Utility of an uncharged coating suppressing EOF enabled further mitigation of non-specific adhesion of proteins to the capillary surface. All major mixture components were identified, and in several cases, separations of charge-based proteoforms from unmodified variants, as well as partial separations of protein complexes from their corresponding monomers were observed. Trastuzumab was then analyzed under native conditions on the same system, resulting in the identification of major glycosylation states of the mAb, including hemiglycosylated species. Moreover, Trastuzumab dimers were identified at approximate abundances of less than 3%. Native analysis revealed partial dissociation of individual chains of the mAb, suggesting that a trace quantity of light chains are bound non-covalently instead of via disulfide bonds; these forms were separated from intact mAb structures in the CZE domain. Finally, the protein extract from E. coli ribosomes was analyzed under native conditions by sheathless CZE-MS using the polyacrylamide-coated capillary coupled to the Q Exactive Plus Orbitrap MS (Thermo Fisher Scientific). As a result, over 90% of the protein groups from E. coli ribosomes were identified in a combinatorial analysis involving native and denaturing conditions. A single analysis under native conditions resulted in 42 identified protein groups, including some 144 proteoforms. Several potential non-covalent complexes between ribosomal proteins were identified by the native approach. Native CZE-MS was demonstrated as a viable technique for the analysis of single protein isolates, such as mAbs, simple mixtures of proteins and protein complexes, and for the protein isolate from an entire cellular organelle the ribosome. This chapter is based on a 2017 publication in the Journal of the American Society for Mass Spectrometry.
CZE-MS offers a complementary approach for the analysis of a variety of protein analytes. In the first investigation, we started with a single protein of 22 kDa with a single glycosylation site. The second investigation involved a more complex protein - a therapeutic mAb candidate - of ~150 kDA and up to two glycosylation sites. Considerable optimization in sample preparation, as well as capillary coatings and analytical conditions was required for the characterization of this analyte. In the third investigation, sample complexity was extended further towards the analysis of protein mixtures and a cellular organelle. This required further method development at all stages of the analytical workflow, beginning with sample preparation, separation and detection parameters, instrumentation, and data analysis strategies. In summary, we have sought to use online CZE-MS in a top-down proteomic format to demonstrate the applicability of this technique for the analysis of a variety of samples of different complexities. Our efforts have resulted in characterization of numerous protein analytes, revealing information concerning these proteins that could not be obtained via more conventional bottom-up proteomic approaches. Lastly, we have demonstrated one of the first examples of the application of online CZE-MS under native conditions.
mass spectrometry
monoclonal antibody
native mass spectrometry
orbitrap
sheathless
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