Multiplexed high-throughput MALDI-MS arrays for optimization of solid phase extraction

• Solid-phase extraction (SPE) is one the most common sample preparation techniques in many laboratories. Optimization of analyte specificity and recovery is crucial for high-sensitivity detection and analysis robustness/reproducibility. The optimization process usually starts with screening for a suitable extraction phase for a certain analyte (e.g. reversed-phase or HILIC) or if the extraction phase is known with the fine tuning of the extraction conditions i.e. loading, washing and elution solutions.This process of optimization can be quite time consuming and expensive, as a solution we here apply our microfabricated proteomic sample handling platform, Integrated Selective Enrichment Target, (ISET) that interfaces MALDI mass spectrometry and is able to rapidly and in parallel handle minute amounts of samples and provide an efficient, economic and generic sample treatment process while allowing for a minimum of sample transfers. This sample preparation device made of silicon, has 96 individual array positions. Each pyramidal array position has an inlet (1000 x 1000 µm) and an array of 9 outlets (20 x 20 µm), defining a volume that can be filled with capture medium, max 0.6 µL of beads. The small bead volume allows for more than 480 (5 x 96) samples to be subjected to SPE on the ISET platform using only of 20 mg stationary phase, i.e. less than the amount provided in an ordinary 1 mL SPE cartridge. At the same time the parallelization provided (96 samples/plate) facilitates multiplexed testing of different loading, washing and elution conditions with enough replicates to ensure the validity of the developed SPE protocol. E.g. using different solutions column-wise with respect to different SPE materials row-wise in the ISET arrays enables easy, high-throughput and very economic multiplex analysis.The utility of the multiplexed MALDI MS SPE optimization scheme is demonstrated using different molecularly imprinted polymers (MIPs) for the selective enrichment of phospho serine (pS) and phospho tyrosine (pY) peptides respectively from a peptide mixture. MIPs are synthetic polymer materials finding increased use in SPE utilizing their specific cavities that provides selective target recognition. With these MIPs materials as a model SPE sorbent study, some 500 samples where run using different loading, washing and elution conditions in triplicate. The data provided an optimized protocol maximizing recovery and specificity while minimizing background. In addition the data from the multiplexed and multiparametric experiments could also be used to draw conclusions on the nature of the MIP – Analyte interactions.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Medicinteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Medical Engineering (hsv//eng)

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