title: On-Chip Mesoporous Functionalized Magnetic Microspheres for Protein Sequencing by Extended Bottom-up Mass Spectrometry.
abstract: A limited amount and extreme concentration variability of proteomic-related samples require efficient analyte preconcentration and purification prior to the mass spectrometry (MS)-based analysis. Preferably, these steps should be coupled online with chosen fractionation and detection techniques for the minimization of the sample loss. To realize such sample pretreatment, herein, an on-chip solid-phase extraction-gradient elution-tandem mass spectrometry (SPE-GEMS/MS) is introduced. This technique combines in a microfluidic format online sample preconcentration/purification on SPE sorbent with further fractionation and MS/MS analysis. C8-functionalized mesoporous magnetic microspheres are chosen as a sorbent, spatially confined with an applied magnetic field. They ensure a selective enrichment and analysis of large hydrophobic peptides (2.5-7 kDa), matching the desired mass bin of the extended bottom-up proteomic (eBUP, 3-7 kDa) approach. Within less than 35 min and without additional sample purification, SPE-GEMS/MS provided 66.5% of protein sequence coverage from 75 fmol of BSA tryptic digest. Analysis of only 33 fmol of a single monoclonal antibody, digested with secreted aspartic protease 9 (Sap9) to large peptides, yielded 80% of its sequence coverage. A more complex equimolar mixture of six antibodies (55 fmol each), submitted to Sap9 proteolysis, was also successfully processed by SPE-GEMS/MS, resulting in 50-67% of the total antibody sequence coverage. Importantly, for all antibodies, unique peptides containing complementarity determining regions were detected for both heavy and light chains, leading to a correct identification of mixture components despite their high sequence homology. Moreover, SPE-GEMS/MS microchip and chosen magnetic sorbent are cost-effective and can be produced and operated in a disposable manner. Therefore, the present technique could be potentially suitable for a high throughput sequencing of monoclonal antibodies and rapid eBUP-based structural protein analysis, especially when only a limited sample amount is available.
authors.affiliation: Laboratory of Physical and Analytical Electrochemistry, EPFL Valais, Ecole Polytechnique Fédérale de Lausanne , 1951 Sion, Valais, Switzerland.
authors.forename: Natalia
authors.initials: N
authors.lastname: Gasilova
authors.affiliation: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne , 1015 Lausanne, Vaud, Switzerland.
authors.forename: Kristina
authors.initials: K
authors.lastname: Srzentić
authors.affiliation: Laboratory of Physical and Analytical Electrochemistry, EPFL Valais, Ecole Polytechnique Fédérale de Lausanne , 1951 Sion, Valais, Switzerland.
authors.forename: Liang
authors.initials: L
authors.lastname: Qiao
authors.affiliation: Department of Chemistry and Institute of Biomedical Sciences, Fudan University , Shanghai 200433, PR China.
authors.forename: Baohong
authors.initials: B
authors.lastname: Liu
authors.affiliation: Centre d'Immunologie Pierre Fabre, 74160 St. Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne, Vaud, Switzerland.
authors.forename: Yury O
authors.initials: YO
authors.lastname: Tsybin
authors.affiliation: Laboratory of Physical and Analytical Electrochemistry, EPFL Valais, Ecole Polytechnique Fédérale de Lausanne , 1951 Sion, Valais, Switzerland.
authors.forename: Hubert H
authors.initials: HH
authors.lastname: Girault
mesh_terms: D060526:Magnetic Fields; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D008863:Microspheres; D010316:Particle Size; D010455:Peptides / Q000032:analysis*; D016062:Porosity; D040901:Proteomics; D020539:Sequence Analysis, Protein / Q000379:methods*; D012822:Silicon Dioxide / Q000737:chemistry*; D052616:Solid Phase Extraction; D013499:Surface Properties; D053719:Tandem Mass Spectrometry
chemical_list: D058185:Magnetite Nanoparticles; D010455:Peptides; D012822:Silicon Dioxide