Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry.

Functional proteomics provides a powerful method for monitoring global molecular responses following activation of signal transduction pathways, reporting altered protein posttranslational modification and expression. Here we combine functional proteomics with selective activation and inhibition of MKK1/2, in order to identify cellular targets regulated by the MKK/ERK cascade. Twenty-five targets of this signaling pathway were identified, of which only five were previously characterized as MKK/ERK effectors. The remaining targets suggest novel roles for this signaling cascade in cellular processes of nuclear transport, nucleotide excision repair, nucleosome assembly, membrane trafficking, and cytoskeletal regulation. This study represents an application of functional proteomics toward identifying regulated targets of a discrete signal transduction pathway and demonstrates the utility of this discovery-based strategy in elucidating novel MAP kinase pathway effectors.

Matrix-assisted laser desorption ionization/quadrupole ion trap mass spectrometry of peptides. Application to the localization of phosphorylation sites on the P protein from Sendai virus.

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.

Mixture analysis using a quadrupole mass filter/quadrupole ion trap mass spectrometer.

A hybrid tandem mass spectrometer is constructed by interfacing a quadrupole mass filter (Q) to a quadrupole ion trap mass spectrometer (QITMS) and is evaluated for the analysis of mixtures. The mass filter is set to selectively inject ions of a particular m/z or, in scanning mode, to sequentially inject ions into the QITMS for subsequent manipulation and detection. Performance of the instrument is demonstrated using a mixture of ions created by electron impact ionization of perfluorotributylamine (FC-43) and peptide ions generated by pulsed Cs+ bombardment. Resulting data are compared to those obtained by utilizing only the ion trap. Molecular weight, fragmentation, and high-resolution analyses for the sequentially injected mass-filtered peptides show improved performance over similar measurements employing only the ion trap mass spectrometer. Performance is optimized when ions are not rf-isolated in the QITMS. Using the hybrid, a resolution of 33,200 is achieved for angiotensin I. Dramatic reduction of space charge-induced signal suppression is demonstrated for LSIMS of Glu-fibrinopeptide B. 'On-the-fly' collision-induced dissociation is performed for m/z 502 from FC-43, where fragmentation is induced by increasing the ion injection energy. Collision-induced dissociation efficiencies for fragmentation of angiotensin I by resonance excitation are investigated as a function of cooling time for different modes of operation of the hybrid. A current limitation of the instrument is the time required to port the data for acquisition.