Matrix-assisted Laser Desorption Ionization/Quadrupole Ion Trap Mass Spectrometry of Peptides

Abstract

The polymerase-associated phosphoprotein (P protein) from Sendai virus, a murine Paramyxovirus, is reported in the literature to be a highly phosphorylated protein. In vitro studies have detected phosphorylation in different regions of the protein, while a single phosphopeptide (identified as the sole phosphorylation) site) was observed using in vivo techniques. In this work, two phosphorylation sites of the P protein from Sendai virus are localized by a direct approach using matrix-assisted laser desorption ionization/quadrupole ion trap mass spectrometry. A computer-aided approach is used to confirm peptide identification.Sendai virus is the murine prototype of the Paramyxovirus, belonging to the order of Mononegalvirales. Related human pathogens include types of parainfluenza virus, mumps, measles, and respiratory syncytial virus as well as the more distantly related Filoviridae viruses Marburg and Ebola. Animal pathogens comprise Newcastle disease, cattle and bird parainfluenza viruses, canine distemper, and murine pneumonia. An understanding of the functioning of the Sendai virus prototype would provide insights into the function and replication of these ubiquitous human and animal viruses.The genome of the Sendai virus consists of a single strand of RNA with negative polarity that codes for at least six structural and five nonstructural proteins (1-3). The RNA core is encapsidated by a helical nucleocapsid protein (NP). Virions enter cells directly through surface membranes, and viral replication and transcription (mediated by viral polymerase) begin immediately in the cytoplasm. Polymerase activity is carried out by the polymerase-associated phosphoprotein (P protein, 1 M r ϭ 65,000) and the large protein, L protein, (M r ϭ 200,000) (4, 5). Almost all of the viral proteins are phosphorylated, however, the P protein appears to be more heavily phosphorylated on a mol/mol basis (6, 7). The P protein also seems to be modular in nature (8). N-terminal and C-terminal domains are conserved among Paramyxoviruses (52% and 69% homology, respectively), while a 100-residue region in the middle is variable with ϳ11% homology (9, 10). The C-terminal domain has been shown to stabilize the L protein (11), and the N-terminal region interacts with the NP protein and has been shown to be essential for RNA encapsidation as well as RNA synthesis (12).A number of studies were undertaken to locate sites of posttranslational modification in order to understand the role that phosphorylation plays in the function of the P protein.In vitro experiments produced conflicting results where phosphorylation sites were detected in the first N-terminal quarter of the protein (7) or in the second N-terminal quarter of the protein (13). More recent work (14) showed that cell-free phosphorylation using virion-associated protein kinase as a phosphorylating agent caused phosphorylation of both serine and threonine. In contrast, intracellular experiments in which the phosphorylation state of the P protein was analyzed during virus replication in...

An atmospheric pressure matrix‐assisted laser desorption/ionization ion trap with enhanced sensitivity

“…Qin and et al 13. used preferential fragmentation at the C‐terminus of Asp and Glu residues as markers to help validate sequence assignments, while Jonscher and Yates14 were able to localize phosphorylated residues using fragment ions inadvertently generated in the source. Although source fragmentation has provided useful information, improved determination of post‐translational modifications may be possible by capitalizing instead on the MS n capability of ion traps.…”

Structural analysis of oligosaccharides by atmospheric pressure matrix‐assisted laser desorption/ionisation quadrupole ion trap mass spectrometry

“…As a consequence, under typical operating conditions, only a single stage of tandem mass spectrometry may be available using these techniques, limiting their scope for the analysis of complex oligosaccharides. In contrast, it has been demonstrated that the quadrupole ion trap is a useful tool for the analysis of biomolecules such as peptides,31–33 oligonucleotides34,, 35 and oligosaccharides,13,, 14 through the use of successive stages of trapping and CAD to reveal structural details that cannot be resolved by MS/MS alone.…”

Analytical methodologies for the detection and structural characterization of phosphorylated proteins

“…Phosphopeptides have also been detected and assigned for modification sites in the course of vacuum or atmospheric pressure MALDI-ion trap-MS experiments [145,148]. In addition to pSer and pThr residues, unexpected elimination of the elements of H 3 PO 4 from pTyr-containing phosphopeptides was also observed under atomospheric pressure ionization conditions [149].…”