A microchip emitter for solid phase extraction – gradient elution – mass spectrometry

A microchip electrospray emitter with a magnetic bead trap has been designed for solid phase extraction-gradient elution-mass spectrometry (SPE-GEMS). The goal of this method is the detection of analytes at low concentrations and it is here demonstrated using reverse phase coated magnetic beads (Mbs) for the preconcentration and detection of the peptides. The sample is passed through the chip, and the peptides are retained and enriched in the trap. After washing, the peptides are released sequentially by stepwise gradient elution and electrosprayed for mass spectrometry analysis. This approach allows effective sample desalting, enrichment, sequential elution and MS detection without the introduction of an additional separation step after SPE. Efficient preconcentration of model peptides by SPE and sequential release and analysis of peptides by GEMS were demonstrated for diluted sample solutions within the range of 1 µM to 10 nM. Fortified human blood serum, protein digest and fractions collected after protein digest OFFGEL separation were analysed by SPE-GEMS allowing the detection of low abundance peptides usually not observed by direct mass spectrometry analysis. A mathematical model for gradient elution is proposed.

Middle-down approaches for mass spectrometry-based protein identification and characterization

Kristina Srzentic

Mass spectrometry (MS) has emerged over the last two decades as the analytical technique of choice in systems-level protein studies, known as proteomics. Two are the MS-based approaches generally applied to proteomics: bottom-up (BU), which relies on the proteolytic digestion of proteins into short (~10 amino acids) peptides, and top-down (TD), where proteolysis is omitted, intact proteins are detected and fragmented in gas phase. Both methods present advantages as well as drawbacks. Here, we sought to establish a complete platform to put forward a third MS-based proteomic approach; middle-down (MD). It implies protein digestion as in BU, but aims to generate large peptides which size approaches the one of small intact proteins that are readily analyzed in TD. This novel domain aims to account for the shortcoming of both classical approaches. Until now, the main reasons behind the limited use of MD proteomics have been the lack of easy-to-use cleaving agents capable of producing peptides in the desired 3-15 kDa mass range with high specificity, limitations in MS and allied instrumentation, and the absence of dedicated bioinformatics tools for processing of acquired data. The latter greatly impedes the next milestone in MD proteomics â large-scale analysis. MD can potentially combine the analysis of large portions of proteins carrying set of biologically-relevant modifications â allowing exploring proteins of molecular weight or complexity incompatible with current TD capabilities â with the high-throughput hallmark of BU proteomics. Here, we first in silico evaluated the potential target amino acid residues to produce peptides in the MD mass range within the proteomes of different organisms. This bioinformatics work was followed by an experimental study based on synthetic MD-sized peptides, aimed at determining the optimal MS and tandem MS parameters for large peptide characterization. Next, we pursued two distinct ways of performing MD proteolysis: i) the use of an enzyme and ii) the use of a chemical reagent. We selected the protease Sap9 as a target enzyme for MD, which we fully characterized and successfully applied to the study of a mixture of monoclonal antibodies, where it showed an advantage over traditional BU in terms of reduced introduction of artifacts to the sample, allowing the post-translational modification investigation and unambiguous antibody identification. The chemical cleavage way we addressed via judicious protocol optimization for hydrolysis at the N-terminal side of cysteine with NTCB reagent. We also advanced MD protocols by generation of large (~50 kDa) subunits of monoclonal antibodies through the use of papain and another more specific novel protease, GingisKHAN, combined with new MS signal processing and data analysis capabilities. The developed workflow improved mapping of the connectivity of cysteines involved in inter- and intra-molecular disulfide bridges in antibodies. To summarize, we demonstrated that MD approach to mass spectrometry and proteomics is a powerful, yet underdeveloped, complement to BU and TD. This work has benchmarked MD for targeted protein analysis. In the near future, with advancements of the field, we envision its growing use for large-scale complex proteome analysis.

EPFL2016

Structural Analysis of Complex Molecular Systems by High-Resolution and Tandem Mass Spectrometry

Yury Tsybin

In this chapter, we describe the advances in high-resolution and tandem mass spectrometry applied for structural analysis of complex molecular systems. Focus is made on Fourier transform mass spectrometers (FTMS) because of their ability to routinely provide resolving power (resolution), which is above 100 000, and correspondingly high mass accuracy, which is higher than 2 ppm. The advantages of using these analytical capabilities have been demonstrated for analysis of extremely complex mixtures of molecules of diverse nature and size. What are the recent and envisioned steps in FTMS method and technique development? What are the novel application areas that await these developments? We describe the state-of-the-art in ion cyclotron resonance (ICR) and Orbitrap FTMS and showcase the advantages of FTMS applications for petroleomics and intact protein mass measurements. We briefly overview the current methods of ion activation and dissociation as most widely employed in tandem mass spectrometry (MS/MS) for peptide and protein structure analysis. Finally, we discuss the analytical limitations of the state-of-the-art FTMS and offer a brief introduction to some of the possible solutions to these limitations. Particularly, we describe the achieved and envisioned benefits of advanced signal processing methods aimed specifically at FTMS improvement.

Wiley-VCH Verlag GmbH & Col. KGaA2014

On-Chip Spyhole Nanoelectrospray Ionization Mass Spectrometry for Sensitive Biomarker Detection in Small Volumes

Hubert Girault, Liang Qiao, Géraldine Margaretha Alice Stauffer, Xiaoqin Zhong

A polyimide microfluidic chip with a microhole emitter (Ø 10–12 μm) created on top of a microchannel by scanning laser ablation has been designed for nanoelectrospray ionization (spyhole-nanoESI) to couple microfluidics with mass spectrometry. The spyhole-nanoESI showed higher sensitivity compared to standard ESI and microESI from the end of the microchannel. The limits of detection (LOD) for peptide with the spyhole-nanoESI MS reached 50 pM, which was 600 times lower than that with standard ESI. The present microchip emitter allows the analysis of small volumes of samples. As an example, a small cell lung cancer biomarker, neuron-specific enolase (NSE), was detected by monitoring the transition of its unique peptide with the spyhole-nanoESI MS/MS. NSE at 0.2 nM could be well identified with a signal to noise ratio (S/N) of 50, and thereby its LOD was estimated to be 12 pM. The potential application of the spyhole-nanoESI MS/MS in cancer diagnosis was further demonstrated with the successful detection of 2 nMNSE from1 μL of human serum. Before the detection, the serumsample spiked with NSE was first depleted with immune spin column, then desalted by centrifugal filter device, and finally digested by trypsin, without any other complicated preparation steps. The concentration matched the real condition of clinical samples. In addition, the microchips can be disposable to avoid any cross contamination. The present technique provides a highly efficient way to couple microfluidics with MS, which brings additional values to various microfluidics and MS-based analysis.

2018

Middle-down approaches for mass spectrometry-based protein identification and characterization

Kristina Srzentic

EPFL2016