10/20/11 1 Lecture 20 – Oct 24 Chem 4101 – Fall 2011 Analytical problem – Mass Spectrometry Table Analyte characteristics 1 Gases, small volatile molecules (includes alcohols) 2 Elemental ions or inorganic complexes 3 Polymers and proteins 4 Mid-size hydrophobic molecules (> 250 Daltons) 5 Mid-size hydrophilic molecules (> 250 Daltons) • Find a table that best fits your analytical problem • Select one analyte at each table. Find its nominal molecular weight (or atomic weight) and the exact mass. • What type of mass spectrometry would be suitable to obtain a spectrum of your analyte? Draw a hypothetical spectrum on the white board. • Random people will explain the answers to the rest of the class Lecture 20 – Oct 24 Chem 4101 – Fall 2011 Mass Spectrometry 1- Identification of compounds 2- Quantification of elements and their ionic species 3- Quantification of organic molecules and biomolecules 4 – Use of MS in your analytical problem -Sections 20D Suggested exercises: 20-18, 20-17, analytical problem examples presented by students Lecture 20 – Oct 24 Chem 4101 – Fall 2011 Quadrupole!Ion Trap!Time-of Flight!Time-of Flight Reflectron!Magnetic Sector!FTMS!QTOF!Accuracy!0.01% !(100 ppm)!.01% !(100 ppm)!.0005%!(5 ppm)!.0002%!(2 ppm)!<.0001 %!(< 1 ppm)!<.0001%!(< 1 ppm)!.0002%!(2 ppm)!Resolution!4,000!4,000!10,000!15,000!30,000!100,000!40,000!m/z range!4,000!4,000!>300,000!10,000!10,000!10,000!10,000!Scan Speed!1 s!1 s!msec!msec!1 s!1 s!1 s!Tandem MS!MS2!MSn!MS!MS2!MS2!MSn!MS2!Tandem MS Comments!Low energy!Low energy!N/A!High energy!Accuracy/ resolution of product ions!Accuracy/resolution sensitivity!General Comments!Low cost, pos/neg switching!MSn!Low cost!Size!Size, expense, high field magnet, high vacuum!High sensitivity and accuracy!Comparison of Mass Analyzers Courtesy of Joe Dalluge10/20/11 2 Lecture 20 – Oct 24 Chem 4101 – Fall 2011 Identification of Compounds • Mass spectrometers are commonly hyphenated with separation techniques (GC, HPLC, CE). The separation reduces sample complexity. The mass spectrometer functions as a detector. • Hyphenation is not necessary if the expected spectrum is simple. For example when it has only one component or several components that are not fragmented. • Multiple components can be identified in a single mass spectrum. • Typical identification strategies include: • Molecular masses from mass spectra. (M+1)+, (M-1)+ • Molecular formulas from exact molecular masses. • Purine (C5H4N4), M = 120.044 Daltons • Benzamidine (C7H8N2), M = 120.069 Daltons • Molecular formulas from isotopic ratios • Structural information from fragmentation patterns • Electron impact ionization. Losses of CH2 groups (Figure 20-26) • MS/MS (Collision induced dissociation). Peptide sequencing Lecture 20 – Oct 24 Chem 4101 – Fall 2011 Quantitative mass spectrometry • Calibration curve using internal standard • Analyte homolog • Isotopically labeled compound (2H, 13C, 15N, 18O) • If the mass spectrometer is hyphenated with a separation technique, each compound can be monitored separately using an extracted ion current. 11-deoxycortisol Internal standard (from Cambridge Isotopes) 11-deoxycortisol (21, 21-D2, 96%) 258.31 Daltons M+ Lecture 20 – Oct 24 Chem 4101 – Fall 2011 Selected ion monitoring (SIM) J. Mass Spectrom. (2009), 44, 12, 1637-1660 MS of unlabeled and labeled beta-tubulin peptide Peptide results from treating protein with trypsin Internal standard has a mass of M+3 The ions are doubly charged (isotopic distribution) What m/z values correspond to the internal standard? MS/MS of peptide Fragment ions for sequencing of the peptide Extracted ion chromatogram (XIC) of peptide and internal standard Quantitation10/20/11 3 Lecture 20 – Oct 24 Chem 4101 – Fall 2011 LTQ-Orbitrap LTQ-FT or Precursor Ion Scan (Orbitrap) MS/MS spectra (LTQ) Nano - HPLC Stable isotope labeling of amino acids in cell culture
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