Gas-Phase Fragmentation Behavior of Oxidized Prenyl Peptides by CID and ETD Tandem Mass Spectrometry.

Farnesylation and geranylgeranylation are the two types of prenyl modification of proteins. Prenylated peptides are highly hydrophobic and their abundances in biological samples are low. In this report, we studied the oxidized prenylated peptides by electrospray ionization mass spectrometry and identified them by collision-induced dissociation (CID) and electron-transfer dissociation (ETD) tandem mass spectrometry. Modified prenyl peptides were generated utilizing strong and low strength oxidizing agents to selectively oxidize and epoxidize cysteine sulfur and prenyl side chain. We selected three peptides with prenyl motifs and synthesized their prenylated versions. The detailed characteristic fragmentations of oxidized and epoxidized farnesylated and geranylgeranylated peptides were studied side by side with two popular fragmentation techniques. CID and ETD mass spectrometry clearly distinguished the modified version of these peptides. ETD mass spectrometry provided sequence information of the highly labile modified prenyl peptides and showed different characteristic fragmentations compared with CID. A detailed fragmentation of modified geranylgeranylated peptides was compared by CID and ETD mass spectrometry for the first time. Graphical Abstract ᅟ.

Mass spectrometry cleavable strategy for identification and differentiation of prenylated peptides.

Prenylation of protein (farnesylation and geranylgeranylation) is involved in several human cancers, such as pancreatic, colon, and acute myeloid leukemia as well as Hutchinson-Gilford progeria syndrome (HGPS), a genetic disease that is associated with premature aging for children. Current biochemical methods are not very efficient in identifying and differentiating large-scale prenylations in vivo or in vitro. There are limited methods available for large-scale detection of prenylated proteins using mass spectrometry and no methods currently available which can distinguish farnesylation and geranylgeranylation modification in a single experimental setup. In this study, a simple and novel method for detection and distinction of large-scale prenylated peptides using mass spectrometry-cleavable approaches was developed. The method utilizes simple chemistry on the prenyl group and cleavable properties of a sulfoxide group in the gas phase to produce a signature mass spectrum during tandem mass spectrometric events. The characteristic masses lost from the modified prenylated peptides distinguished the types of prenylation. We also introduced epoxy groups in the prenylation sites of the proteins to make them more hydrophilic and enrichable from complex samples. Stability of the epoxide group was also studied under liquid chromatography-mass spectrometry (LC-MS) conditions. The proof-of-concept of this method was established using prenylated peptides which mimicked the prenyl motifs in the proteins. We believe this method will advance the identification and differentiation of the types of prenylation in proteins in large-scale studies and will improve significantly our knowledge of the mechanism of cancer, cancer treatments, and diagnosis.

Cavity-enhanced absorption measurements across broad absorbance and reflectivity ranges.

Cavity-enhanced spectrometry constitutes an important and highly sensitive technique for absorbance measurements. The current practice generally involves very high reflectivity mirrors and hence intense light sources (typically lasers) to have enough light transmitted. Available theory describes the situation only for high-finesse cavities (high-reflectance mirrors) and generally for systems with very low absorbances. We develop the general expression for absorbance regardless of mirror reflectivity or the absorbance and show that in the limit of high reflectivities and low absorbances it predicts the same numerical values as that derived by O'Keefe (Chem. Phys. Lett. 1998, 293, 331-336; Chem. Phys. Lett. 1999, 307, 343-349). Signal to noise in any photometric system is also dependent on the amount of light reaching the detector because of shot noise limitations. We show that a small aperture in the entrance mirror greatly improves light throughput without significant departure from the theoretically predicted amplification of absorbance; such simple modifications result in real improvement of detection limits, even with mirrors of modest reflectivity and inexpensive detectors. This allows the merits of cavity enhancement measurements to be demonstrated for pedagogic purposes.