On-Chip Mesoporous Functionalized Magnetic Microspheres for Protein Sequencing by Extended Bottom-up Mass Spectrometry
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- 作者：Natalia Gasilova ; Kristina Srzenti? ; Liang Qiao ; Baohong Liu ; Alain Beck ; Yury O. Tsybin ; Hubert H. Girault
- 刊名：Analytical Chemistry
- 出版年：2016
- 出版时间：February 2, 2016
- 年：2016
- 卷：88
- 期：3
- 页码：1775-1784
- 全文大小：440K
- ISSN：1520-6882
文摘
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.