QTOF Small Molecule Applications Update. April 10 th, David A. Weil. Application Scientist, Schaumburg, IL

Transcription

1 QTOF Small Molecule Applications Update April 10 th, 2008 David A. Weil Application Scientist, Schaumburg, IL December 6 th, 2007

2 Agenda Goal Share Best Practices, Updates Software/Hardware Communication Past Recorded Webex s available on website Recorded Video s on use of MassHunter Qual and MassHunter Quant QTOF User Sponsored Group (QTOF@googlegroups.com ) Today s Topics: Care and Feeding of QTOF/TOF Differential and Impurity Profiling Overview of MassHunter MassProfiler Software Overview of Personal Metlin Database Software Questions and Answer Period

3 Care and Feeding of QTOF/TOF Defragment Hard Drives (Weekly) Use Diskeeper to Defragment Drives If Errors Run Check Disk for Errors Program Check LC Components Firmware If older version update LC Firmware

4 Care and Feeding of QTOF/TOF (Cont.) Software Updates MassHunter Qual and Protein Decovolution Patch 1 MassHunter Quant (B.01.04) MassHunter Qual (B.01.03) Patch 1 Metabolite ID B New April 2008 Mass Profiler B METLIN Personal Metabolite Database B.01.01

5 Mass Profiling A Powerful Workflow Mass Profiling: is used to find differences between sample sets originally developed for proteomics/metabolomics can be applied to many analytical problems where finding differences between samples is important is done using statistical analysis Molecular Feature: a discrete molecular entity defined by the combination of RT, mass and response in an LC/MS or GC/MS analysis typically represents a known or unknown compound LC/MS or GC/MS Analysis GC/MS LC/MS Find molecula r features MFE (LC/MS) AMDIS (GC/MS) Compare sample sets Mass Profiler GeneSpring MS Identification Spectrum Mill Molecular Formulas DB Search (METLIN) Library Search Validation QQQ MRM GeneSpring MS

6 Molecular Feature Extractor (MFE) How it works Map signals in the 3-dim. space in time and mass at the MS level. IMPORTANT: High resolution in mass and time! Remove areas which only contain noise, and NO signals. Identify all mass signals with a common RT (narrow time window) Combine mass signals with common RT and chemical relation (isotope, adduct, dimer, higher charge state) => Molecular feature or Compound. Create Extracted Compound Chromatograms (ECC) and Compound Mass Spectra from ions associated to a molecular feature (compound) Compound list or Molecular feature list RT m/z Abund

7 MassProfiler Dependent on MFE Feature Extraction Don t extract all the compounds only Important ones How to Mine for Gold Generated a Base Peak Chromatogram Input Mass Range of Compounds Exclude Reference Ions Input m/z to 4 decimal places Set window to 20 ppm Run Standards to Determine RT Information

8 Good Quality *.MHD Gives Good Quality Profiler 1. Centroid versus Profile data 2. S/N versus Total Counts Typically use 100 counts Too high may not detect C13 peak Profile S/N 10 First Pass 3. Focused MHD generation Limit Retention Time Limit m/z Range 4. Ion Species (NH 4 If buffer) 5. Check Box if High Salt 6. Check if Free Radicals/Quats December 6 th, 2007

9 Good Quality *.MHD Gives Good Quality Profiler 7. Charge State = 1 to detect only singly charged species 8. Last Tabs only for Display in MassHunter QUAL 9. *.ProPos from Profile 10. *.CentPos from Centroid

10 Find Compounds by Molecular Feature

11 NEW Software Mass Profiler B G3297AA Identify similarities and differences in features across multiple analyses/samples from *.MHD data files generated from MassHunter Qualitative Analysis Software Aligns and normalizes features (RT, m/z and intensity) Identify composition of features from Molecular Formula Generation (MFG) and AMRT database searching Compatible with Agilent TOF and QTOF Products Includes Example Data Files, Quick Start Guide Minimal Statistic in Mass Profiler (Student t test) therefore best results with at least 3 replicates of Good/Bad sample and a blank run Cost $3,150

12 Mass Profiling Software Processed Processed TIC Processed TIC Group TIC A (Impure) Aligns Data RT, Mass, Abundance Processed Processed TIC Processed TIC Group B TIC (Control) What s Changed? Mass/RT >2 Fold Change What s All Unique!! Data RT/Mass Impurity Control

13 Mass Profiler B Highlights Impurity Profiling/Differential Analysis Software Graphical Displays Mass vs Retention Time Log2 Ratio (Group1/Group2) vs Retention Time Log2 Abundance Group 2 vs Log2 Abundance Group 1 Unique to Group 1 vs RT and Unique to Group 2 vs RT (New) Feature Identification (NEW) Molecular Formula Generation and AMRT database Single Feature or Batch Processing (summary report) Web Internet Database Searching

14 Graphical Displays

15 Mass Profiler Reproducibility of Results Reproducible Retention Time Stability Spread in ChromatographyRT Spread of Peak in Time

16 Mass Profiler Reproducibility of Results Outlier Sample or Feature

17 Blue Unique Group 2 and Red Unique Group 1

18 Mass Profiler Examples

19 Pharmaceuticals Personal Care Products in Water Problem: Solution: ID and Profile Changes in Chemical Residues Found in Streams Throughout North America RRLC/QTOF with Exact Mass Chemical Residue Mass Profiler ID Differences Between Streams Critical Component: Automated Data Analysis.. 20, Features Outcome: Ability to ID ppb level Chemical Residues in Water December 6 th, 2007

20 Pharmaceutical Personal Care Products in Water QTOF MFE/Profiling Experiment (44 Compounds) Neutral Masses of Potential Residues in Water Acetaminophen Cotinine Aspirin ,7-dimethylxanthine Caffeine Thiabendazole Clofibic acid Naproxen Carbamaepine Busproprion Albuterol Gemifrozil Cimetidine Sulfamethoxazole Ketoporfeb Diphenhydramine Venlafaxine Sulfamethizole Trimethoprim Sulfamethazine Sulfachloropyridazine Triclosan Norsertraline Diclofenav Norfluoxetine HCTZ Duloxetine Codeine Sertraline Wararin Fluxetine Sulfadimethoxine Triclocarban Ranitidine Fluvoxamine Citalopram Paroxetine Furosemide Dehydroifedipine Enalaprilat Miconazole Diltiazem simvastatil Erythromycin

21 Mass Profiling > 20,000 features S/N >2 RI 0.001% M ass vs. Retention Tim e Mass R eten tion T ime (min ) Group 4 vs 10 December 6th, 2007

22 Inclusion List Utilized to Reduce Display +/ daltons 450 Mass vs. Retention Time Mass Retention Time (min) Selecting any point on graph gives detailed MW, Empirical Formula info

23 RT 9.63 C 15 H 12 N 2 O Carbamaepine > 5X Higher in Sample 10 > 4

24 Impurity Analysis: Drug Delivery Product Extractable Materials in Saline Solutions Objective: Identify Molecular Weight, Formula Structural Information of Impurities Method: RRLC, MFE/Profiler, Top 50 for Targeted MS/MS Results: > 30 Compounds ID, mass error

25 Comparison Sample/Control BPC at 500 µl/min x BPC Scan SampleAutoMSMS_New0002.d 1 1 Sample x BPC Scan ControlAutoMSMS_New2.d 1 1 Control Counts vs. Acquisition Time (min)

26 Mass Profiler to ID Differences: Sample/Control 515 Features, RI > 0.01%, in all replicates

27 174 Unique Features in Sample 88 Unique Features in Control

28 C 16 H 19 N 3 SO 5 Degradation Products of Amoxicillin and AmoxClavin MW NH 2 Source Tablet 875mg New Tablet 500mg 09/07 Sample Preparation HO O NH O N S OH CH 3 CH 3 O Tablets Dissolved in 40mL Methanol Water with 0.1% Formic Acid Syndicated 20 minutes Spin 5 minutes RPM Dilute 100 and 1000 fold with water December 6 th, 2007

29 Degradation of Amoxicillin S/N 5:1, RI > 0.01%, Mass , RT 1-11 Minutes 835 Features

30 Amoxicillin Degradation Products Isotope Pattern, Background Subtraction, # Ions

31 Features only in Degraded Drug

32 Composition and Database Searching

33 Plant Extract with 16 Spiked Components

34 Plant Extract 16 Compounds Scaling Factor Total Conc Changes Rat input/output

35 Plant Extract 16 Compounds

36 Mass Profiler- How to Use

37 Mass Profiler The Basics Double Click Mass Profiler Icon 1 for Single Group Comparison 2 for Differential Analysis

38 Mass Profiler Load MHD Files *.MHD Data File Location Select Add then add in the *.MHD files in Groups Holding Down ctrl key select all *.MHD to add Then Select OK

39 Set Up Mass Profiler Method *.mpm is Extension of Mass Profiler Project

40 Mass Profiler Edit Method 1. Limit Mass Range and RT 2. For Small Molecule Apps Multiply Charge Forbidden 3. For Biomolecules Apps Multiply Charged Required 4. Abundance to 0.1 to 0.01% 5. Number of Ions >=2 6. Isotope Pattern Matching

41 Mass Profiler How to Import Exclusion List Sort Table By Height/Volume Right Click on Table Select EXPORT From EXCEL Copy Masses From Table

42 Mass Profiler Exclusion List of Masses Paste List of Masses From Blank Into Exclude Masses

43 Mass Profiler Alignment

44 Mass Profiler Results Filters

45 Mass Profiler Special Case No Replicates For Example without Replicates Min Differential Score MUST be set to ZERO before loading *.MHD Files

46 Feature Identification Set Parameters for MFG

47 Identify Composition of Features Right Click any feature in Graph Right Click any feature in Table

48 Individual Feature Results # Compositions and Database Hits

49 Identification Summary Report

50 Feature Details

51 Exact Mass/Formula Database Searching

52 Export Feature Information

53 Questions and Answers

54 METLIN Personal Metabolite Database Software

55 METLIN Personal Database G6825AA Metabolite-specific database for metabolomics research Database installed on a PC Contains ~15000 compounds Manual and batch searches Query based on monoisotopic mass Customizable Add compounds Assign chromatographic retention times to metabolites Create subset databases Works with other Agilent software Cost: $3759

56 METLIN Personal Database Search Capabilities Manual searches Search on neutral or charged monoisotopic mass User-specified search tolerance Optionally restrict by retention time Molecular formula Compound name Retention time (user defined) Batch and manual mass searches from Agilent products GeneSpring MS MassHunter Qual Mass Profiler

57 METLIN Personal Database Initial View User search mode

58 METLIN Manual Single Mass Search Single mass search settings Active metabolite structure Hyperlinks to Metabolite websites Metabolite search results

59 METLIN Batch Search Load file for batch search Active metabolite structure Active mass Active mass search results

60 METLIN Batch Summary Active metabolite structure Batch summary best metabolite match Exportable list of best metabolite search results

61 Creating a New METLIN Database METLIN is read only and can not be changed Create a new personal database from METLIN

62 Edit Metabolite Database Update Entry Add retention time to existing metabolite

63 METLIN Mass Search With Retention Time Adds Specificity Only 1 metabolite instead of 3!!!! Adding retention time eliminates isobaric metabolites

64 Edit Metabolite Database New Entry Select add new then type new metabolite information

65 METLIN Search from Mass Hunter Qual Identify compounds: search database Specify the METLIN database Set search parameters Manual search options Select ion in spectra Select MFE compound(s) Batch search Automate by Find and Identify Find compounds by MFE Search database Generate formula (optional) Name and CAS numberdatabase and MFG results Set Parameters View detailed results

66 METLIN Searching From Mass Profiler Mass Profiler METLIN searching is virtually the same as METLIN Mass Profiler can optionally add MFG MFG is used to assist METLIN search METLIN results are exportable Database customization is done only in METLIN Retention time changes New compounds New database METLIN search integrated into Mass Profiler

67 Summary Care and Feeding of QTOF/TOF Remember to Defrag Hard Drives Weekly Software updates available Mass Profiler B $3,150 Differential Analysis Software Batch Molecular Formula Generation and Database Searching METLIN Personal Metabolite Database B $3,759 Accurate Mass Retention Time Database (AMRT) Convert *.CSV to MTL Formats User Generated Databases (Plant Extract, Pesticides, AA s)

68 Questions and Answers Thank You

69 Upcoming Proteomics eseminars April 24, 2008, 11:00 a.m. EDT Biomarker Discovery by Targeted and Profiling Proteomics Professor Rainer Bischoff, Analytical Biochemistry, University of Groningen May 8, 2008, 11:00 a.m. EDT Protein Analysis Using CAD/ETD Ion Trap Tandem Mass Spectrometry Professor Ole Norregard Jensen, Protein Research Group at University of Southern Denmark June 26, 2008, 11:00 a.m. EDT Peptide Quantitation With An Agilent 6410 QQQ System Ning Tang, Application Scientist, Agilent Technologies, Inc.