1. Just launched Operated by Karel Harant 1

2. Rapid development of instrumentation Decline of MALDI-based instrumenst 2D protein electrophoresis abandoned Development of many quantification techniques Emploing of data-independent acquisition and quantification Label-free quantification More than half of human proteome from one sample injection 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 acquistion 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. Each type of mass analyser has disadvantages Overcome by combination of analysers into one machine Until summer 2013 two was usual combination Fusion – first tribrid instrument on the market 6

7. Ion trap Fast and sensitive detector Orbitrap Precise and high resolution detector Quadrupole filter 7

8. Orbitrap Fourier transformation type of mass analyser High resolution (450 000) 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) 8

9. 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 9

10. Quantification in MS 2 suffers from ratio flattening Fusion can select multiple precursors in IT from MS 2, fragment them and measure ratios in MS 3 Speed is reduced to one half in comparison with MS 2 approach, but results are better 10

11. We don´t have ETD (electron transfer disociation) Allows effective study of glycosylations Technically we can analyse phosphorylation, ubiquitination and alternative N-terminal sequence 11

12. Regular check of chromatogram profile, intensity and number of IDs in our standart sample and in Thermo Hela digest From 2hours gradient we acquire 4500-5000 IDs with 1%FDR Check (minimally twice per day) peak shape and intensity on Cyt C Thermo standart 12

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14. Gel-based protein identification Simple one by MALDI Tof/Tof (190kč for piece) Complex one on Fusion Complex proteome analysis 4500-5000 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) 14

15. Protein identification and TMT quantification Proteome discoverer Scaffold Label-free quantification Max Quant and Perseus Sieve 15

16. Multiple reaction monitoring via targeted approach Phosphoproteome analysis 16

17. 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 17

18. Comparison of five yeast strains, two cells populations from each. Totaly 10 diffrent samples. 4000 Kč sample preparation Label-free 33750 Kč measurement of samples (60minutes gradient) in triplicate Or TMT 27000 Kč TMT labeling 7500 Kč measurement of sample in duplicate(4 hours gradient) Total price of both approaches under 40000 Kč – 4000 Kč for sample 18

19. 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 19

20. Laboratory of sequencing DNA Faculty of Science, Charles University of Prague YEARNumber of Sequencing Number of Fragment analysis 200910 7008 100 201013 30016 150 201120 65013 050 201222 90013 200 201323 50017 000 201426 9009 500 https://www.natur.cuni.cz/biologie/servisni-laboratore/laborator-sekvenace-dna Founded in 2003. Present – laboratory is self-sufficient including salaries of two employees.

21. High-throughput Next- generation sequencing on MiSeq Illumina Sanger sequencing and fragment analysing on ABI 3500XL genetic analyzer

22. Wikimedia Read length - 800 bp

23. 3500 XL obrázek Template (DNA) Preparation Type of DNA templates sequenced - PCR product, Plasmid, Phage/cosmid, BAC clones etc. After generation, PCR products need to be purified to remove excess primers and 4 dNTPs. Purification can be done either enzymatically, or by passing through a column or from a gel. Other templates, e.g. plasmids are usually prepared in sequence quality grade. Cycle Sequencing Reaction by Sanger's dideoxy Terminator Method on a PCR Machine Components - DNA, primer, heat resistant DNA polymerase, 4 dNTPs, 4 dideoxy terminator nucleotides (4 ddNTPs) fluorescently labeled with 4 different dyes, and enzyme buffer containing Mg++, K+. Fragment Separation by Capillary Electrophoresis on ABI 96- capillary 3730XL Sequencer The samples are electrokinetically injected into the array of capillaries, The negatively charged fragments migrate toward the anode by size, the smallest ones move fastest. Their tagged ddNTP terminators can be read as the fragment's base sequence. A laser beam excites these dye molecules as the fragments reach a detection window producing fluorescent signals that are collected from all 96-capillaries at once, spectrally separated and focused onto a CCD camera. Very sophisticated optical and electronic devices produce a color readout that is translated, with the help of a sequence analysis software, into a sequence, as we see it.

24.  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

25. 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

27. The 2100 Bioanalyzer Agilent is a microfluidics- based platform that is used for the quantification and qualification analysis of DNA, RNA, proteins, and cells.evaluation of RNA, DNA, and protein samples The Covaris M220 Focused-ultrasonicator generate fragment sizes between 150 and 5,000 bp in tight, highly reproducible fragment distributions and is designed for next generation sequencing applications that require high-quality DNA fragmentation for library preparation. Quantification: Accurate quantification of input material is crucial to efficient library construction and sequencing. Quantus Fluorometer, NanoDrop Spectrophotometer