Use of peptides and proteins in the characterization of electrospray ionization and quadrupole ion trap mass spectrometry

Kathleen Ann Cox, Purdue University

Abstract

Several projects are discussed which utilize the unique capabilities of mass spectrometry in the characterization of peptides and proteins. The determination of primary structure can be achieved through the use of an MS/MS experiment in which protonated molecular ions are dissociated into structurally characteristic fragments through collisions with an inert target gas. Evaluation of this collision-induced dissociation (CID) process in a quadrupole ion trap mass spectrometer, indicates a predominance of relatively low energy collisions. Since higher energy collisions will produce more fragments, and thus more detailed sequence information, alternatives to the traditional CID process are explored in an effort to enhance the energy deposition of this process. These alternatives include (i) multiple dissociation stages $(\rm MS\sp{n}),$ (ii) mixed target gases, (iii) boundary activated dissociation (BAD) and (iv) fast DC pulses. Examination by electrospray ionization mass spectrometry indicates that higher order structural information can be obtained for larger protein ions ($>$10 kDa). Low energy collisions in the RF-only collision cell of a triple quadrupole mass spectrometer indicate two distinct charge state distributions. These distributions are proposed to correspond to a more highly charged, open conformational form and a lower charged, folded form. Different cross sectional areas of these two conformers allow preferential selection of one distribution over the other on the basis of ion mobility through the target gas. Although MS/MS spectra produce characteristic fragment ions indicative of peptide structure, the behavior of peptide ions during the dissociation process is not well characterized. Fragmentation patterns are dictated by the amino acid residues present in the peptide as well as their position along the backbone. Recently developed methods to obtain high resolution and mass measurement accuracy have been combined with the inherent sensitivity and $\rm MS\sp{n}$ capabilities of the ion trap in order to elucidate fragmentation mechanisms of various peptides. Finally, high resolution analyses of peptide systems are utilized in the evaluation of complex mass shifts observed in ion trap mass spectra. Although mass measurement accuracies are currently better than 0.01%, a close inspection of the calculated mass errors reported reveals non-random distributions. The nature of these shifts is examined in detail in an effort of overcome the last major obstacle in transforming the ion trap into a high performance mass spectrometer.

Degree

Ph.D.

Advisors

Cooks, Purdue University.

Subject Area

Analytical chemistry

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