Using optimized collision energies and high resolution, high accuracy fragment ion selection to improve glycopeptide detection by precursor ion scanning

Judith Jebanathirajah 0 1 2 Hanno Steen 0 1 2 Peter Roepstorff 0 1 0 Published online June 2, 2003 Address reprint requests to Dr. P. Roepstorff, Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, DK-5230 Odense, Denmark 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark , Odense, Denmark 2 Current address: Department of Cell Biology, Harvard Medical School , 240 Longwood Ave., Boston, MA 02115, USA Glycosylation is the most widespread protein modification and is known to modulate signal transduction and several biologically important interactions. In order to understand and evaluate the biological role of glycosylation it is important to identify the glycosylated protein and localize the site glycosylation under particular biological conditions. To identify glycosylated peptides from simple mixtures, i.e., in-gel digests from single SDS PAGE bands we performed high resolution, high accuracy precursor ion scanning using a quadrupole TOF instrument equipped with the Q2 pulsing function. The high resolving power of the quadrupole TOF instrument results in the selective detection of glycan specific fragment ions minimizing the interference of peptide derived fragment ions with the same nominal mass. Precursor ion scanning has been previously described for these glycan derived ions. However the use of this method has been limited by the low specificity of the method. The analysis using precursor ion scanning can be applied to any peptide mixture from a protein digest without having previous knowledge of the glycosylation of the protein. In addition to the low femtomole (nanomolar) detection limits, this method has the advantage that no prior derivatization or enzymatic treatment of the peptide mixtures is required. (J Am Soc Mass Spectrom 2003, 14, 777-784) 2003 American Society for Mass Spectrometry - Cin a remarkable resource of protein and putative urrent genome sequencing efforts have resulted protein sequences. These protein sequences, which are stored in accessible databases, increase the facility with which proteins may be identified using mass spectrometry. As protein identification at the subpicomole level becomes routine, there is a shift in interest from simple protein identification, i.e., correlation of a sample spot on a PAGE gel with an accession number, to protein characterization with special emphasis on the analysis of the co- and posttranslational protein modifications. One of these modifications is protein glycosylation, which is the most common protein modification. It is estimated that 50% of all proteins are glycosylated [1]. This modification plays a major structural role but is also heavily involved in cell-cell recognition and modulating molecular interaction. Furthermore, there is the increasing notion that reversible glycosylation plays a pivotal role in signaling mechanisms [2]. In order to understand and evaluate the biological role of glycosylation in detail it is important to analyze the glycosylation. A comprehensive analysis comprises three steps, each of them is a challenge on its own: (1) The identification of glycosylated proteins and peptides, (2) the localization of the glycosylation sites, and (3) the elucidation of the glycan structure. One of the major problems with glycosylation analysis is the fact that this modification is normally highly heterogeneous such that it induces a fairly undefined mass shift that can easily exceed the mass of the peptide. In contrast, simple modifications such as phosphorylation or acetylation induce well-defined mass shift (or multiples thereof). One approach that can be used to identify glycosylated peptides is precursor ion scanning for glycan derived fragment ions. As further MS/MS studies of the identified glycopeptides can be performed immediately after their identification, much information about the glycopeptide may be derived. Subsequent mass spectrometric experiments elucidating the sequence or structure of the peptide and the glycan can be executed, in order to localize the glycosylation site. Information with respect to the compositions and the sequences of the glycans can also be obtained. The characteristic fragment ions used for this purpose are normally the reporter oxonium ions of hexose at m/z 163.060, of N-acetylhexosamines at m/z 204.084, and of hexoylhexosamine at m/z 366.139 [3], but much larger oxonium ions have also recently been used [4]. These fragment ions enable the selective detection of glycosylated peptides using precursor ion scanning or skimmer fragmentation routines on triple or single quadrupole mass spectrometers, respectively. However, Carr and coworkers reported a lack of specificity for these precursor ion experiments whereby other nonglycosylated peptides produce signals in the precursor ion scan as the produce other peptide-derived fragment ions that have the same nominal mass as the characteristic reporter ion [5]. This problem is commonly encountered when low resolution triple quadrupole mass spectrometers are used for precursor ion experiments. In order to successfully use a triple quadrupole mass spectrometer for precursor ion scanning experiments characteristic fragment ions have to be unique within 0.5 Da. This factor has prevented the widespread use of precursor ion experiments for protein modification analysis in general and in particular for protein glycosylation analysis, as many characteristic fragment ions are not unique within the required limits. However, in some cases the selectivity of precursor ion experiments can be significantly increased by using high resolution, high accuracy quadrupole TOF mass spectrometers equipped with so-called Q2 pulsing, which enables precursor ion experiments on quadrupole TOF instruments with similar sensitivities as on triple quadrupole mass spectrometers. Using this type of tandem mass spectrometer for precursor ion scanning it is sufficient if the reporter fragment ion is unique within 0.03 Da as compared with the 0.5 Da, required when using the traditional triple quadrupole mass spectrometer. Since the second mass analyzer in quadrupole TOF instrument, the TOF, cannot be used as filter, the acquired spectra are not true precursor ion spectra but reconstructed spectra. This implies that longer scan times are necessary, thereby limiting this approach. The application of this high resolution precursor ion scanning was recently demonstrated by Steen et al. for the selective detection of various protein modifications such as tyrosine phosphorylation and nitration, tryptophan bromination, and proline hydroxylation by utilizing the corresponding immonium ions [6 9]. All the immonium ions used for high resolution, high accuracy precursor ion scanning showed a mass difference less than 0.1 Da with respect to the interfering peptidederived fragment ions with the same nominal masses. This mass shift was adequately large such that the resol (...truncated)