MALDI-TOF Mass Spectrometry and Introduction to Proteomics Dr. Steve Hartson Oklahoma State University Dept. Biochemistry and Molecular Biology Recombinant DNA/Protein Resource Facility

“Since its introduction more than 100 years ago, mass spectrometry has become a ubiquitous research tool. The number of scientific discoveries that MS enabled and the number of Nobel Prizes awarded to scientists who contributed to the method’s evolution is astounding. Used extensively by chemists, biologists, pharmacologists, and scientists in several disciples, MS how holds a prominent spot in the world of analytical techniques.” - Tanuja Kopal Editor in Chief Genomics & Proteomics

Joseph John Thomson 1906 Nobel Prize for Physics "in recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases” Francis William Aston 1922 Nobel Prize for Chemistry "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” Wolfgang Paul 1989 Nobel Prize for Physics "for the development of the ion trap technique” John Bennet Fenn 2002 Nobel Prize for Chemistry "for the development of soft desorption ionisation methods (ESI) for mass spectrometric analyses of biological macromolecules” Koichi Tanaka 2002 Nobel Prize for Chemistry "for the development of soft desorption ionisation methods (MALDI) for mass spectrometric analyses of biological macromolecules" “Astounding prizes!”

MS is a leading biotool Session subjects at the 2004 meeting of the Association of Biomolecular Resource Facilities

OK, how about modern times? In the 2005 ABRF meeting, mass spectrometry and proteomics were given their own satellite meeting on a separate day. In 2006, it was business as usual, with MS and proteomics comprising 45% of the presentations; no other subjects came close.

MALDI-TOF mass spectrometer MALDI Ion source TOF Mass Analyzer

“MALDI-TOF” mass spec M atrix- A ssisted: Your favorite molecule is co- crystallized (dried) with a light-adsorbing compound (“matrix”).

“MALDI-TOF” mass spec Laser Desorption/Ionization : A pulse of laser light is used to force molecules into gas phase and ionize them.

MALDI-TOF mass spectrometry Ions are then accelerated in a high- voltage electrostatic field…

MALDI-TOF mass spectrometry …and allowed to drift down a long high- vacuum flight tube. At the end of the tube, ions strike a detector. Drift time (time of flight) is measured by sophisticated electronics.

Interpreting a mass spectrum x-axis is… –tof –m / z –“mass” of ion y-axis is… –“hits” on the detector –number of ions detected

TOF~velocity~acceleration~mass The relationship of TOF v.s. mass is calibrated via co-analysis of standards whose masses are known.

More spec’s... Exacting determination of masses (20 to100 ppm mass accuracy) Exquisite sensitivity (routine detection sub-pmoles of peptide)

Mass Spec Concepts and Vocab “ions” “mass” “isotopes”

Ions Ion = “charged molecule” Only ions are detected in MS For ionization techniques that are typically used for biological molecules, ions are generated via the ejection or capture of a proton. The mass of a proton is ~1 AMU; the charge of a proton is +1 Ionic mass [MH] +1 versus molecular mass (subtract ~1 from raw data)

Isotopes Stable natural isotopes –extra neutrons versus the periodic table –not radioisotopes

Isotopes For peptide-sized molecules, most mass spec’s can resolve (n) versus (n +1 AMU). The result is that a “single peptide” actually yields a series peaks differing by one AMU.

Overview of a typical protein identification by MALDI-TOF Isolation of protein(s) of interest Cleavage at specific residues MALDI-TOF analysis of resultant peptides Extract peptide masses from the spectrum Search databases for matching “peptide mass fingerprint”

Cleavage Fingerprinting to establish protein identity Cleveland mapping: after cleavage with a specific protease, peptide products are compared in adjacent lanes on an SDS-PAGE gel.

PMF: each MS peak is represents the peptide product of a cleavage.

A “peptide mass” fingerprint "Peaks" represent individual peptides. A "peak list" representing these peptides' masses can be generated and used.

Experimental proteins v.s. Hypothetical proteins Digest experimental protein with protease Size resultant peptides Generate list of “peak masses” (a.k.a., peptide mass fingerprint) Archive of protein sequences “Index” via in silico digestion with protease Calculate theoretical masses of individual “peptides.” Generate a “look up table” for use by a scoring algorithm to allow ranking of “matches.” At this point in the technology, the database is not built from experimental mass spec data!

Logistics Five Units / Four groups Rotation as per page 3 Lecture Mon morn, Thurs morn Thurs & Friday by reservation final Unit: assessing your samples mopping up “Sticking points” Voyager U1 Sample prep (U4B)