Anal. Chem. 1998, 70, 4939-4944
Analysis of Phospho- and Glycopolypeptides with Infrared Matrix-Assisted Laser Desorption and Ionization Rainer Cramer,† Wilhelm J. Richter,†,‡ Elaine Stimson,† and Alma L. Burlingame*,†,§,|
Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, U.K., Department of Pharmaceutical Chemistry, University of California, 513 Parnassus Avenue, San Francisco, California 94143-0446, and Department of Biochemistry, University College of London, Gower Street, London WC1E 6BT, U.K.
The analytical characteristics of infrared (IR) matrixassisted laser desorption and ionization (MALDI) were investigated for the analysis of phosphopeptides, a phosphopolypeptide, and glycopeptides. Two commercially available instruments, a high-resolution delayed extraction (DE) reflectron time-of-flight (RETOF) mass spectrometer and a high-power pulsed Er:YAG laser, were interfaced to produce a high-resolution MALDI-DE-RETOF instrument that is easy to use and can be switched between UVand IR-MALDI mode within seconds. In the interface design, particular attention was paid to maintaining the same professional operating environment for the new IRMALDI mode as exists for the commercial UV-MALDI mode. This instrument configuration facilitates comparative observation and investigation of the relative analytical merits of IR- and UV-MALDI. The results of studies of the tryptic r-casein phosphopeptides, RP1 (a Thr45monophosphorylated congener of the recombinant protein hirudin variant 1), and fetuin Asn81 tryptic glycopeptides are presented. The elimination of labile substituents such as phosphoric acid and sialic acid is suppressed in IRMALDI-RETOF mass spectrometry, with concomitant higher analyte ion yields. These results reflect the advantages that accrue from deposition of significantly less internal energy in the case of IR-MALDI. In the last year, reports of investigations involving infrared matrix-assisted laser desorption and ionization (IR-MALDI) have increased.1-8 Most of these recent IR-MALDI reports deal with fundamentals and the elucidation of basic mechanisms1-4 or the * Corresponding author: (phone) 415/476-5641; (fax) 415/476-0688; (e-mail)
[email protected]. † Ludwig Institute for Cancer Research. ‡ Deceased May 3, 1998. § University of California. | University College of London. (1) Sadegh, M.; Olumee, Z.; Tang, X.; Vertes, A.; Jiang, Z.-X.; Henderson, A. J.; Lee, H. S.; Prasad, C. R. Rapid Commun. Mass Spectrom. 1997, 11, 393. (2) Berkenkamp, S.; Menzel, C.; Karas, M.; Hillenkamp, F. Rapid Commun. Mass Spectrom. 1997, 11, 1399. (3) Niu, S.; Zhang, W.; Chait, B. T. J. Am. Soc. Mass Spectrom. 1998, 9, 1. (4) Caldwell, K. L.; McGarity, D. R.; Murray, K. K. J. Mass Spectrom. 1997, 32, 1374. (5) Eckerskorn, C.; Strupat, K.; Schleuder, D.; Hochstrasser, D.; Sanchez, J. C.; Lottspeich, F.; Hillenkamp, F. Anal. Chem. 1997, 69, 2888. 10.1021/ac9803939 CCC: $15.00 Published on Web 10/24/1998
© 1998 American Chemical Society
advantages of direct analysis of proteins electroblotted from 2DPAGE gels.5-7 However, there are only a few reports concerning the nature of IR-MALDI spectra (e.g., in comparison to UVMALDI) of peptides and proteins bearing labile moieties, even though the detection of these types of biomolecules intact is of considerable importance in establishing the correct composition and stoichiometry. It has been known for some time that metastable decay may be minimized by employing IR- rather than UV-MALDI. Interestingly, the IR-MALDI postsource decay (PSD) mass spectrum of substance P is qualitatively comparable to that observed using UV-MALDI,9 despite the fact that the laser energy deposition per unit volume in IR-MALDI is ∼1 order of magnitude smaller than in UV-MALDI.10 The initial ion velocities for both wavelength ranges appear to be comparable.11 Taken together, these findings appear to be partially contradictory and thus raise questions about the underlying processes and mechanisms involved in IR-MALDI. Irrespective of these points, it is of clear importance to structural biology to investigate the inherent and possibly unique analytical features of IR-MALDI. As reported previously by Berkenkamp et al.,2 the reduced degree of metastable decay provides substantial improvements for the analysis of high-molecular-mass molecules, but there has been no comparable investigation of the analytical benefits of IRMALDI throughout the lower mass range of widespread interest in the characterization of proteolytic digests of proteins. This report presents IR-MALDI data for a polypeptide and components of protein digests in the lower mass range (
