MS training course

Vikas Kumar, PhD "In recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases.“ 1906

Cited from: http://masspec.scripps.edu/mshistory/ First MS called “Parabola spectrograph”

Ion were separated by different parabolic trajectories in electromagnetic field.

Detection by the ions striking a fluorescent screen or photographic plate. Designed a mass spectrometer in which ions were dispersed by mass and focused by velocity--which improved MS resolving power by an order of magnitude over the resolution Thomson had been able to achieve. Aston’s design was the basis of his later instruments with which he systematically and accurately measured the masses of the isotopes of many of the elements.

Francis William Aston

1922 Nobel Prize "For his discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive elements, and for his enunciation of the whole-number rule."

Cited from: http://masspec.scripps.edu/mshistory/ In 1920 A. J. Dempster developed single focusing magnetic deflection instrument.

He also developed the first impact source, which ionizes volatilized molecules with a beam of from a hot wire filament.

In 1940s Alfred O. C. Nier developed sector field MS. It was used during World War II to do isotopic analysis, with separation of uranium-235 from uranium-238.

The Calutron, a three-story-high version of Nier's magnetic sector instrument, separated uranium-235 for the first atomic bomb.

Cited from: http://masspec.scripps.edu/mshistory/ In 1946 W. E. Stephen from University of Pennsylvania proposed TOF in that ions are separated by differences in their velocities as they move in a straight path toward a collector in order of increasing mass-to-charge ratio.

Improved by W. C. Wiley and I. H. McLaren from Bendix Corp., Detroit, Mich in mid 1950s and further improved by invention of reflectron B. A. Mamyrin in 1974.

When commercial TOF instruments first came out their performance in resolution was so poor that they never lived up to even single-focusing magnetic instruments. However, this analyzer has been greatly improved recently to almost match the most sophisticated, and very expensive Ion cyclotron resonance MS.

Cited from: http://masspec.scripps.edu/mshistory/

Quandrupole was first reported in the mid-1950s by of the University of Bonn. Then Paul also developed quadrupole ion Wolfgang Paul trap, which can trap and mass-analyze ions using a three-dimensional quadrupolar radiofrequency electric field. An ion trap system was first introduced

Hans Georg Dehmelt commercially in 1983 by Finnigan MAT.

“For the development of the ion trap technique.” 1989 Nobel prize Cited from: http://masspec.scripps.edu/mshistory/

1956 Gas Chromatography (GC/MS) 1956 Identifying Organic Compounds with Mass Spectrometry 1962 Mass Spectrometry Imaging 1966 Chemical Ionization 1966 Peptide Sequencing 1966 Tandem Mass Spectrometry 1966 Metabolomics 1968 Electrospray Ionization 1968 Collision Induced Dissociation

Cited from: http://masspec.scripps.edu/mshistory/

1974 Fourier Transform Ion Cyclotron Resonance 1974 Extra-Terrestrial Mass Spectrometry 1975 Atmospheric Pressure Chemical Ionization (APCI) 1978 Triple Quadrupole Mass Analyzer 1980 Inductively Coupled Plasma MS 1984 Quadrupole/Time-Of-Flight Mass Analyzer 1987 Soft Laser Desorption of Proteins 1989 Monitoring Enzyme Reactions with ESI-MS

Cited from: http://masspec.scripps.edu/mshistory/

1990 Protein Conformational Changes with ESI-MS 1990 Clinical Mass Spectrometry 1991 MALDI Post-Source Decay 1991 Non-covalent Interactions with ESI 1992 Low Level Peptide Analysis 1993 Oligonucleotide Ladder Sequencing 1993 Protein Mass Mapping 1996 Intact Virus Analyses

Cited from: http://masspec.scripps.edu/mshistory/

Two recently developed MS techniques have had a major impact on the ability to use MS for the study of large biomolecules: electrospray ionization MS (ESI MS) and ESI matrix-assisted laser desorption/ionization MS (MALDI MS).

ESI was first conceived in the 1960s by Malcolm Dole of John B. Fenn Northwestern University, Evanston, but it was put into practice in the early 1980s by John B. Fenn of Yale University.

MALDI MS, a form of laser desorption MS, was developed in MALDI 1985 at the University of Frankfurt, Germany, by Franz Hillenkamp, and independently by Koichi Tanaka and coworkers at Shimadzu Corp., Kyoto, Japan. Koichi Tanaka "For the development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules.“ 2002 Nobel prize Cited from: http://masspec.scripps.edu/mshistory/

1998 Electron Capture Dissociation (ECD) 1999 Quantitative Proteomics and Metabolomics with Isotope Labels 2000 Orbitrap 2004 Electron Transfer Dissociation (ETD) 2005 Direct Analysis in Real Time (DART) 2014 Draft of Human Proteome

Cited from: http://masspec.scripps.edu/mshistory/ Mass spectrometry is a powerful analytical technique that is used to  Identify unknown compounds.  Quantify known compounds.  Elucidate the structure and chemical properties of molecules.

Highly sensitive, It can detect compounds in very minute quantities (femtomoles).

Mass Spectrometer (Smallest scale) A mass spectrometer is an instrument that measures the masses of individual molecules that have been converted to ions; i.e., molecules that have been electrically charged.

Ionization Mass Sorting (filtering) Detection

Ion Ion Source Mass Analyzer Detector

Form ions Sort Ions by Mass (m/z) (charged molecules) Detect ions

100

75 Inlet • Solid 50 • Liquid 25 0 • Vapor 1330 1340 1350 Mass Spectrum

Atmospheric Low Pressure (~10-6 torr) Pressure (760 torr)

Direct Insertion: Done using insertion plate/probe. The sample is placed onto a probe having loading chamber separated from ionization region of the mass spectrometer through a vacuum interlock.

Direct Infusion: A simple capillary or a capillary column is used to introduce a sample as a gas or in solution. Direct infusion is also useful because it can efficiently introduce small quantities of sample into a mass spectrometer without compromising the vacuum. Capillary columns are routinely used to interface separation techniques with the ionization source of a mass spectrometer. Protonation: In this method proton is added to a molecule, producing a net positive charge of 1+ for every proton added. M + H+ MH+

Positive charges tend to reside on the more basic residues of the molecule, such as amines, to form stable cations. MALDI, ESI and APCI use this approach. Some compounds are not stable to protonation (i.e. carbohydrates) or cannot accept a proton easily (i.e. hydrocarbons). Deprotonation: in this method net negative charge is achieved through the removal of a proton from a molecule. M - H+ M-H-

This mechanism of ionization, commonly achieved via MALDI, ESI, and APCI is very useful for acidic species including phenols, carboxylic acids, and sulfonic acids. This method is compound specific. Cationization: in this positively charged ion is produced by non-covalently adding a positively charged ion to a neutral molecule. M + C+ MC+ Although similar to protonation however it refers to addition of a cation adduct other than a proton (e.g. alkali, ammonium). Cationization is commonly achieved via MALDI, ESI, and APCI. Carbohydrates are excellent candidates for this ionization mechanism, with Na+ a common cation adduct. Limited information in MS/MS Electron ejection: In this method ionization through the ejection of an electron to produce a 1+ net positive charge, often forming radical cations. M -e- M+

Observed most commonly with electron ionization (EI) sources, electron ejection is usually performed on relatively nonpolar compounds with low molecular weights and it is also known to generate significant fragment ions. Often generates too much fragmentation, it can be unclear whether the highest mass ion is the molecular ion or a fragment.

Electron capture: In this method a net negative charge of 1- is achieved with the absorption or capture of an electron. M +e- M-

It is a mechanism of ionization primarily observed for molecules with a high electron affinity, such as halogenated compounds. Often generates too much fragmentation, it can be unclear whether the highest mass ion is the molecular ion or a fragment.

Ionization Source Acronym Event

Electrospray ionization ESI evaporation of charged droplets

Nanoelectrospray ionization nanoESI evaporation of charged droplets

Atmospheric pressure chemical ionization APCI corona discharge and proton transfer

Matrix- assisted laser desorption/ionization MALDI photon absorption/proton transfer

Fast atom/ion bombardment FAB ion desorption/proton transfer

Electron ionization EI electron beam/electron transfer

Chemical ionization CI proton transfer

• A mass spectrometer does not measure mass, it measures the mass-to-charge ratio (m/z), z must not be zero. • Ionization can be achieved by adding or removing a charged particle. • A mass spectrum is an intensity vs. m/z plot representing a chemical analysis. • Peak intensity is NOT a linear response to the abundance because of the differences in ionization and detection efficiencies. It is also energy dependent. The most intense peak is called the base peak and is arbitrarily assigned the relative abundance of 100 %.

F: FTMS + c NSI Full ms [300.00-1000.00] 421.76 100

90

80

450.25 70

60

50 529.81 40 674.86

Relative Abundance Relative 30 540.80 353.54 20 523.29 842.51 10 652.85 310.83 396.24 465.74 574.80 610.34 685.85 743.44 803.11 899.82 930.48 993.65 1058.61 0 300 400 500 600 700 800 900 1000 m/z

Atomic weight of an element is a weighted average of the naturally occurring isotopes. Resolution: How well separated are the peaks from each other?

Mass accuracy: How accurate is the mass measurement?

Sensitivity: How small an amount can be analyzed?

High resolution means better mass accuracy

Resolution =18100 8000 15 ppm error

6000 Resolution = 14200 24 ppm error 4000 Counts

Resolution = 4500 2000 55 ppm error

0

2840 2845 2850 2855 Mass (m/z) A) Thomson’s MS 15 B) Dempster’s MS 100 C) Aston’s third MS 2000

MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information.

Mixture of Single Fragments ions ion

Ion MS-1 MS-2 source One precursor selected for MS/MS

+ MS/MS + + + +

Have only masses The masses of all to start the pieces give an MS/MS spectrum

One atomic mass unit (A.M.U or u) = 1 Dalton (Da) = 1.66 x 10-24 g

Small Molecule: 2 ~ 2,000 Da. Protein: 500 ~ 10,000,000 Da Human Chromosome: 109 ~ 1011 Da Human Genome: 2 x 1012 Da Microbial Genome: 109 ~ 1010 Da Nanoparticle: 104 ~ 1012 Da Microparticle: 1011 ~ 1022 Da Virus: ~ 109 Da Cell: 1013 ~ 1015 Da

2.5ug/ul in Spray mix 1:1 ACN:WATER 0.5%FA UE511 05-Feb-201419:32:56 140205_NAINITA_CYCLIC_1 91 (7.840) AM (Cen,4, 80.00, Ar,10000.0,0.00,1.00); Sm (SG, 5x10.00); Sb (2,40.00 ); Cm (86:181) A3;752.0723 100 2253.19±0.00

752.4142

%

752.7581

753.1003

770.0656 753.4277 770.7365 750.6907 753.7507 765.0775 771.7452 730.5913 733.4211 740.6890 746.7525 782.1030 m/z 0 730 735 740 745 750 755 760 765 770 775 780 785 790 795 800 805 810 815 820

With sufficient accuracy, unique molecular formula can be determine

e.g. Distinguish CO, N2, CH2N and C2H4 (all having m/z 28)

12C 12.0000 14 CO N2 N 14.0031 16O 15.9949 14N 14.0031 27.9949 28.0062

12 12 CH2N C 12.0000 C x2 24.0000 1 C2H4 H x2 2.0156 1H x4 4.0312 14 N 14.0031 28.0312 28.0187 250 fmoles in Spray mix 1:1 ACN:WATER 0.5%FA UE511 05-Feb-201416:51:02 140205_MYO_2 44 (3.788) AM (Cen,4, 80.00, Ar,10000.0,0.00,1.00); Sm (SG, 2x10.00); Sb (2,40.00 ); Cm (31:71) A19;893.2102 100 16951.86±0.05 A20 848.5995

A21 808.2383

A17 998.1753 A22 771.5474 250 fmol/µl of A23 Myoglobin (16951.49Da) 738.0452 in Spray Mix

A16 (Detected with 21.82 ppm 675.7334 1060.4969 % error)

A15 1131.1282

616.2368 A14 1211.8521

A13 1304.9928

A12 1413.6598 A11 1542.0876

1218.8632 1312.5446 A10 543.4824 1421.8362 1551.0085 1696.1947 1225.8722 1320.0989 1430.0153 1706.0156 1583.4767 1884.5657 1942.2233 0 m/z 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

10 relative quantification Replicate LC limited to ID-limited to producingfeatures -real (ANOVA)statistics -very sensitive -observe differencesacross states -Quantitatefeatures across replicates Label-Free LCMS Retention Time MS of samples yieldsof samples -MS

60 of peptides

m/z

RT State 1 State m/z

DifferentialOverlay Mapping Aligned,Normalized LC 10 State 2 State m/z Retention Time

State 3 State m/z -MS 60

400 2000 m/z MS

100 556.62 556.95

50 557.29 554.13 555.66 557.60

553.60 Abundance Relative 554.30 557.94 0 552 554 556 558 560 m/z • Finding environmental toxins: GC/MS systems are ideal for identifying pesticides, gasoline oxygenates, and algal toxins. The newer LC/MS-MS systems are more sensitive and are capable of deeper analysis of water soluble organics such as surfactants, explosives, and pharmaceuticals. • Testing for Steroid Use in Athletes: GC/MS technology is especially effective in confirming the presence or absence of steroids, diuretics, and stimulants in a person’s body. • Fighting Terrorism: The Mass Spectrometry Toxin Laboratory can detect botulinum toxins, anthrax, and ricin in a given sample and is refining state-of-the-art mass spectrometry technology to “fingerprint” the toxin in a way that can help identify the source of the agent. • Drug Development and Discovery: Drug developers and scientists rely on mass spectrometry to characterize chemical structures and identify impurities within their compounds. Pharmaceutical researchers often use mass spectrometry with liquid chromatography technology (LC/MS) to obtain quantitative data for the evaluation and submissions process necessary for FDA approval. • In astronomy for analyses of the components of our solar system, for all geochronology. • In chemistry for chemical analysis and for identification of complex natural products and of metabolic pathways. • In materials analysis and process monitoring in the petroleum, chemical, and pharmaceutical industries, and they are being used in food processing and electronics industries.

Magnetic sector mass analyzer Sample ovens and ion source Detector

Martian Exospheric Neutral Composition Analyzer – MENCA

Quadarpole MS in Indian Mars mission http://www.thermo.com/com/cda/resources/resources_detail/1,2166,200553,00.html Journal of the American Society for Mass Spectrometry Rapid Communications in Mass Spectrometry Journal of Mass Spectrometry International Journal of Mass Spectrometry European Journal of Mass Spectrometry Mass Spectrometry Reviews Analytical Chemistry Journal of the American Chemical Society Proceedings of the National Academy of Sciences Journal of Chromatography Journal of Proteome Research Electrophoresis Journal of Biological Chemistry http://www.ionsource.com http://www.asms.org/whatisms/index.html http://mass-spec.lsu.edu/msterms/index.html http://masspec.scripps.edu/mshistory/whatisms_details.php http://ca.expasy.org