Just launched. Operated by. Karel Harant

Transcription

1 Just launched Operated by Karel Harant 1

2 Rapid development of instrumentation Decline of MALDI-based instruments 2D protein electrophoresis abandoned 80% of proteomic data acquired in last two years (Somebody, HUPO2014) 2

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4 Tens of thousands MS 2 spectra from 1hour gradient Computational processing Most comon approach Generation of theoretical mass list Compare real precursor MS 1 mass with theoretical precursor mass If there is match - compare theoretical and real MS 2 spectra No sequence reading Full sequence information rarely obtained You need to know the genomic sequence of organism of your interest 4

5 Sensitive Acquire low abudance peptides Short acquisition times for high abudance peptides Fast More spectras = more identifications Acurate For effective data processing and confidence of your IDs High resolving power Resolve nearly isobaric precursors Integration of more fragmentation approaches ETD for PTM analysis Allow higher order of MS n fragmentation Presence of ion trap Perform reporter ion quantification in MS 3 5

6 Orbitrap Fourier transformation type of mass analyser High resolution ( ) and accuracy (sub ppm) Qadrupole Mass filter Fast and precise precursor selection Prevent filling of downstream mass analysers with unwanted ions Ion trap is most sensitive type of mass analyser One charge give detectable signal Combination with Qudrupole for fast ion selection Trap filled only with desired ions Faster and more sensitive Have limited capacity 20Hz operating frequency (proteomic application) 6

7 Majority of methods - only relative quantification Targeted approach allows to measure exact concentrations Four basic principes Labeled in MS 1 SILAC metabolic labeling during cultivation Dimethyl labeling labeling after digestion Labeled at MS 2 level - detection of reporter ions TMT ITRAQ Label-free DIA-based methods SWATH 7

8 Regular check of chromatogram profile, intensity and number of IDs in our standart sample and in Thermo Hela digest From 2hours gradient we acquire IDs with 1%FDR Check (minimally twice per day) peak shape and intensity on Cyt C Thermo standart 8

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10 Gel-based protein identification Simple one by MALDI Tof/Tof (190kč for piece) Complex one on Fusion Complex proteome analysis IDs from 120min gradient (2,5 hours of instrument time) from in house prepared human cell lysate, 4900 IDs from 120min gradient of Thermo Hela digest Including sample preparation Detergent (SDC) lysis Trypsin cleavage and sample desalting Relative quantification of samples (not all IDs are quantified) Up to 10 samples with TMT Unlimited number with label-free approach SILAC samples (SILAC labeling must be done by customer) 10

11 We dont want co-authorship of publications, our services are charged Prices set to cover staff, maintenance and consumables Not definitive, will be revisited after 6 months of facility run Costs of laboratory maintenance (energy, services) is not yet included 750 Kč for one hour of instrument time including operator s time and basic data analysis 2500 Kč for TMT labeling of one sample 300 Kč for one hour of operator s time (data analysis) 400 Kč sample preparation (FASP detergent method) 250kč consumables 150kč operator s time 11

12 Comparison of five yeast strains, two cell populations from each. 10 diffrent samples in total Kč sample preparation Label-free Kč measurement of samples (60minutes gradient) in triplicate Or TMT Kč TMT labeling 7500 Kč measurement of sample in duplicate(4 hours gradient) Total price of both approaches under Kč 4000 Kč for sample 12

13 Basic quantification based on three best peaks (top three), not really reliable 800 Kč sample preparation 3000 Kč for two 60min gradients Label-free quantification - reliable 800 Kč sample preparation 9000 Kč for 6 1H gradients 600 Kč data analysis 13

14 Laboratory of sequencing DNA Faculty of Science, Charles University of Prague Founded in Present laboratory is self-sufficient including salaries of two employees. YEAR Number of Sequencing Number of Fragment analysis

15 Sanger sequencing and fragment analysing on ABI 3500XL genetic analyzer High-throughput Nextgeneration sequencing on MiSeq Illumina

16 Sequencing reaction Price: 100 CZK/sample Read length bp More than 200 samples/ day Capillary electrophoresis ABI 3500XL - 24 capillaries Source Wikimedia

17 Price: 40 CZK/sample Our standard: plus 20 Czk/sample Microsatellite analysis AFLP, RFLP SNP genotyping Calibration for 4-dye, 5-dye and 6-dye samples: DS-30 (6FAM, HEX, NED, ROX ) - standard ROX 400, 500 DS-33 (6FAM, VIC, NED, PET, LIZ ) - standard LIZ 500, 600 DS-02 (dr110, dr6g, dtamra, drox, LIZ ) - standard LIZ 120 DS-36 (6FAM, VIC, NED,SID,TAZ,LIZ ) - standard LIZ 500, 600

18 Amplicon sequencing 16S Metagenomics Small genome de novo sequencing Small genome reseq. indel, SNPs RNA sequencing gene expresion, transcriptom sequencing, smallrna seq. Sequencing library quality control

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20 Library preparation price depends on the method and incoming material (DNA, RNA) Examples of price per run MiSeq Reagent Kit Read Length Total Time* Output Price of kit Machine time price 150 v3 2 x 75 bp ~20 hrs Gb CZK CZK 300 v bp ~24 hrs Gb CZK CZK 500 v bp ~39 hrs Gb CZK CZK 600 v3 2 x 300 bp ~55 hrs Gb CZK CZK *includes cluster generation, sequencing and analysis 20

21 The Core Facility offers not only services but also access to all instruments. Quality and quantity of input material are crucial to efficient library construction and sequencing. The 2100 Bioanalyzer Agilent system provides sizing, quantitation and quality control of DNA, RNA and proteins. The Covaris M220 Focused-ultrasonicator generates fragment sizes between 150 and 5,000 bp in tight, highly reproducible fragment distributions. NanoDrop Spectrophotometer Quantus Fluorometer