MALDI Mass Spectrometry: A Label-Free Solution for Ultra-High-Throughput Screening
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1 MALDI Mass Spectrometry: A Label-Free Solution for Ultra-High-Throughput Screening MIPTEC Conference 2016 Meike Hamester, Jens Fuchser Bruker Daltonik, Bremen 1
2 MALDI PharmaPulse for: Biochemical Screening Enzyme small molecule interactions Fluorescence label Fluorescence label > compounds Typically fluorescence-based technologies are used for HTS/ultra HTS in Drug Discovery Typical HTS campaigns have 1-3% hit rates of which a significant proportion may be false positives or assay artefacts Numerous sections need to be performed to reduce hits down to manageable numbers risk of leaving the real hits behind Lengthy hit qualification occurs at the end of a screen to find the true hits using label-free or biophysical techniques Why Mass Spectrometry? Highly sensitive technique Direct quantification of substrate and product (label free) Eliminates need to develop specific antibodies or fluorescent labels Decreased risk of interference Fast assay development Low cost per well
3 MALDI TOF Basic principle of MALDI-Time-of-Flight mass analysis
4 Mass Spectrometry in Drug Discovery Hit Identification Primary HTS (Millions) Fluorescence Hit Confirmation 10,000s MALDI MS SPE MS Dose Response Confirmation (100 s) Hit Characterisation (hit to candidate) SAR/Selectivity Screening (100 s) Mechanism of Action/Kinetics (10 s) Off target effects/safety pharmacology (DMPK) (100 s) Multiplex LC MS/MS
5 Typical Applications of MALDI-TOF rapiflex MALDI TOF/TOF
6 rapiflex MALDI TOF Mass Spectrometry From Imaging to uhts 2 khz 10 khz Laser Smartbeam technology: HTS benefits from all those enhancements: Parallelization of the processes Laser/stage movement ( Run and Shoot ) & data processing Nd:YAG Laser Nd:YAG Laser: Smartbeam Cleaning: no high throughput with frequent cleaning
7 MALDI MS Imaging rapiflex (TOF/TOF) Drug Distribution in rat lung tissue 4mg CHCA matrix, sublimation 20µm pixel resolution, positions, 400 laser shots/position, Analysis time: 41 mins Samples courtesy of Peter Marshall, GSK 7
8 MALDI MS Imaging - rapiflex (TOF/TOF) Drug Distribution in rat lung tissue 4mg CHCA matrix, sublimation (Bruker Home-built device) 5µm pixel resolution, positions, 400 laser shots/position, Analysis time: 36 mins Samples courtesy of Peter Marshall, GSK 8
9 Speed -> huge throughput rapiflex: designed to accelarate imaging However, it meets all requirements of uhts Robustness -> constant operation Definition -> superior image quality 30-Sep samples on MPP Sample Plate
10 Typical Applications of MALDI-TOF Challenges to apply MALDI to HTS and uhts Fully automatic sample plate change every 8-10 min Fully automatic sample preparation ( spotting ) Robustness to analyze 2-3 mio samples without intervention Fit for purpose software package Cost per sample
11 Automation Fully automatic MALDI TOF uhts Courtesy of : CyBio / Analytik Jena 11
12 MALDI Sample Plate Preparation LABCYTE ECHO CyBio CyBi-Well 1536 Analytik Jena 30-Sep-16 12
13 MALDI TOF: Correlation with SPE Mass Spectrometry R=0,83 JMJD2c R=0,95 Acetylcholinesterase Courtesy of Melanie Leveridge (GSK Stevenage, UK)
14 NEW DUB Assay for MALDI TOF Courtesy of: Matthias Trost, University of Dundee
15 Assay transformation TR-Fret MALDI LC-MS SPE-MS
16 Assay Development Support for MALDI 384 MALDI Plate with different assay preparations and acquisition conditions 16
17 A How Can we Further Boost Throughput? Beyond B Increasing spot density from 1536 to 6144 per target can save 5 minutes per 1536 well plate ~ 100 hours over a 2 million compound screening campaign 1 7
18 The Boost for Higher Throughput New laser technology enables up to 20X speed compared to existing instruments We have demonstrated read times of 9.2 minutes/6144 spotted target and 7.3 minutes/1536 spotted target for the ACHe assay.
19 Summary The development of a 10 khz MALDI-TOF instrument (rapiflex) allows label free High Throughput Screening Automation of the rapiflex MALDI TOF fulfils the requirements for uhts and can be used in primary screening campaigns MS for primary screens Label-free 1536 well plate format 6144 possible Sample time: rapiflex: sec 100 x less solvent (& waste) compared to SPE/ LC-MS Low cost (2-3 cent per well) Sample volume nl multiple sampling per well time course studies
20 Thank you! 20
21 True 64-bit application: improved handling of FTMS datasets Skyline projection: shows highest intensity mass signal in dataset, making signals present only in small areas easy to find Integrated Spatial Segmentation using Hierarchal Clustering Rapid classification of up to 50,000 spectra without the need for exporting to external programs 21
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