Low energy peptide fragmentations in an ESI-Q-Tof type mass spectrometer.

Efficient peptide sequencing relies on both high quality MS/MS data acquisition and exhaustive knowledge of gas-phase dissociation mechanisms. We report our contribution to the elaboration of more comprehensive fragmentation models required for efficient automated MS/MS spectra interpretation. Following a statistical approach, various peptides (296 sequences of variable compositions and lengths) were prepared and subjected to low-energy collision-induced dissociations (CID) in an electrospray hybrid instrument (ESI-Q-q-Tof type mass spectrometer) that has retained relatively limited attention so far. Besides, our studies were focused on low molecular weight singly charged peptides that often failed to be identified by sequencing algorithms. Only half of the studied compounds showed charge directed dissociations in accordance with the mobile proton model producing fragment ions directly related to the primary sequence. For the peptides that did not exhibit the expected fragment ion series, alternative dissociation behaviors issued from complex rearrangements were evidenced.

Tandem mass spectrometry of amidated peptides.

The behavior of C-terminal amidated and carboxylated peptides upon low-energy collision-induced dissociation (CID) was investigated. Two sets of 76 sequences of variable amino acid compositions and lengths were synthesized as model compounds. In most cases, C-terminal amidated peptides were found to produce, upon CID, an abundant loss of ammonia from the protonated molecules. To validate such MS/MS signatures, the studied peptides contained amino acids that can potentially release ammonia from their side chains, such as asparagine, glutamine, tryptophan, lysine and arginine. Arginine, and to a lesser extent lysine, was shown to induce a competitive fragmentation leading to the loss of ammonia from their side chains, thus interfering with the targeted backbone neutral release. However, when arginine or lysine was located at the C-terminal position mimicking a tryptic digest, losses of ammonia from the arginine side chain and from the peptide backbone were completely suppressed. Such results were discussed in the frame of peptidomic or proteomic studies in an attempt to reveal the presence of C-terminal amidated peptides or proteins.

MALDI-TOF MS analysis of soluble PEG based multi-step synthetic reaction mixtures with automated detection of reaction failure.

Macromolecules of tunable solubility, used to mimic inert insoluble materials while maintaining solution conditions, allowed the performance of efficient supported organic chemistry and facilitated in situ reaction monitoring. To satisfy the high throughput requirements of automated synthetic processes, organic syntheses carried out on bifunctional polyethylene glycol polymers (PEG(3400)-OH) were monitored step-by-step by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A protocol was designed to control the ionization mechanism of such polymers exhibiting high affinity for alkali metal cations. Automated, rapid, and reliable data interpretation was performed by an in-house developed visual basic application relying on the sodiated ion accurate monoisotopic mass measurement. The methodology was illustrated through the monitoring of a six-step synthetic scheme.

Automated monitoring of phosphatidylcholine biosyntheses in Plasmodium falciparum by electrospray ionization mass spectrometry through stable isotope labeling experiments.

The metabolic pathways contributing to phosphatidylcholine biosyntheses in Plasmodium falciparum, the malaria-causing parasite, was explored by electrospray ionization mass spectrometry. Phosphatidylcholine produced by the CDP-choline pathway and by the methylation of phosphatidylethanolamine was identified and quantified through isotopic labeling experiments. A straightforward method based on cone voltage directed in-source fragmentations and relative abundance measurement of endogenous versus deuterated specific fragment ions was developed for simple and rapid automated data acquisition. Such high-throughput analytical protocol allowed us to measure the relative contribution of two different metabolic pathways leading to phosphatidylcholine without performing technically more demanding and time-consuming MS/MS or LC/MS experiments.

Electrospray mass spectrometry of 2-aminotroponimines and 2-aminotropones.

A series of aminotroponimine and aminotropone derivatives were studied by electrospray mass spectrometry. MS and MS/MS data were acquired according to automated procedures. In the case of mono- and di-halogenated compounds, their specific behavior upon collisionally-activated dissociation conditions provided rapid structural assignments through inexpensive isotopic labeling MS/MS experiments.

Imaging combinatorial libraries by mass spectrometry: from peptide to organic-supported syntheses.

Supported peptide and drug-like organic molecule libraries were profiled in single nondestructive imaging static secondary ion mass spectrometric experiments. The selective rupture of the bond linking the compound and the insoluble polymeric support (resin) produced ions that were characteristic of the anchored molecules, thus allowing unambiguous resin bead assignment. Very high sensitivity and specificity were obtained with such a direct analytical method, which avoids the chemical release of the molecules from the support. Libraries issued from either mix-and-split or parallel solid-phase organic syntheses were profiled, demonstrating the usefulness of such a technique for characterization and optimization during combinatorial library development. Moreover, the fact that the control was effected at the bead level whatever the structure and quantity of the anchored molecules allows the sole identification of active beads selected from on-bead screening. Under such circumstances, the time-consuming whole-library characterization could thus be suppressed, enhancing the throughput of the analytical process.

New example of proline-induced fragmentation in electrospray ionization mass spectrometry of peptides.

The positive ion electrospray ionization (ESI+) mass spectra of peptides usually display only protonated molecules provided that soft ionization conditions are applied (low cone voltage to prevent in-source dissociations). Such ions can be multiply charged depending on the molecular weight of the studied compounds. We have experienced an unexpected behavior during the ESI analysis of a modified peptide of relatively high mass (3079 Da). A specific fragmentation occurred even under soft energetic conditions, leading to a mass spectrum containing multiply charged molecular and fragment ions. The selective rupture involved the amide bond between the glutamic acid and proline residues (E-P sequence). The successive replacement of each amino acid by an alanine residue (positional scanning study) was undertaken to assess which part of the sequence induced such selective and abundant fragmentation on multiply charged species. The succession P-P was evidenced as the minimum unit giving rise to the first peptide bond rupture in the sequence X-P-P. Any acidic amino acid at the X position (X = D, E) favored the fragmentation by an intramolecular interaction. Such proline-induced fragmentation occurring readily in the source differed from the literature data on the specific behavior of proline-containing peptides where bond ruptures occur solely in dissociation conditions.