In-depth LC–MS-based proteomic
profiling of limited biological
and clinical samples, such as rare cells or tissue sections from laser
capture microdissection or microneedle biopsies, has been problematic
due, in large, to the inefficiency of sample preparation and attendant
sample losses. To address this issue, we developed on-microsolid-phase
extraction tip (OmSET)-based sample preparation for limited biological
samples. OmSET is simple, efficient, reproducible, and scalable and
is a widely accessible method for processing ∼200 to 10,000
cells. The developed method benefits from minimal sample processing
volumes (1–3 μL) and conducting all sample processing
steps on-membrane within a single microreactor. We first assessed
the feasibility of using micro-SPE tips for nanogram-level amounts
of tryptic peptides, minimized the number of required sample handling
steps, and reduced the hands-on time. We then evaluated the capability
of OmSET for quantitative analysis of low numbers of human monocytes.
Reliable and reproducible label-free quantitation results were obtained
with excellent correlations between protein abundances and the amounts
of starting material (R
2 = 0.93) and pairwise
correlations between sample processing replicates (R
2 = 0.95) along with the identification of approximately
300, 1800, and 2000 protein groups from injected ∼10, 100,
and 500 cell equivalents, resulting from processing approximately
200, 2000, and 10,000 cells, respectively.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of β-secretase (BACE1), a key enzyme of the progression of Alzheimer's disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method's applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research. Graphical abstract ᅟ.
Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β‐secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β‐secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.
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