Atmospheric Pressure Matrix-assisted Laser Desorption/ionization Mass Spectrometry Research Articles

Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits.

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.

Recent developments in atmospheric pressure MALDI mass spectrometry

A new atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) ion source that includes an extended capillary for delivery of AP-MALDI ions toward a mass spectrometer (MS) atmospheric pressure interface inlet aperture, an optical fiber for laser energy transfer to a target, and x– y travel stages for high throughput analysis of multiple samples, is described. The new source has been interfaced with a Thermo Finnigan LCQ ion trap mass spectrometer bringing powerful high throughput tandem MS capabilities to MALDI analysis. The resulting AP-MALDI-LCQ instrument demonstrated an absolute sensitivity in the low femtomole range for simple peptide mixture analysis, a mass range up to 2000–4000 (limited by LCQ), and an extensive fragmentation of singly-charged AP-MALDI ions in MS/MS experiments. The potential strength of this technique has been illustrated by numerous applications ranging from fragile molecule analysis to tryptic digest analysis for protein identification.

Identification of tyrosine sulfation in Conus pennaceus conotoxins alpha-PnIA and alpha-PnIB: further investigation of labile sulfo- and phosphopeptides by electrospray, matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry.

Liquid chromatography/electrospray ionization mass spectrometry was used to investigate the peptide composition of the venom of Conus pennaceus, a molluscivorous cone shell from the Red Sea. Based on observed M(r)s, this venom contained all known conotoxins previously isolated and identified from this species. Interestingly, the doubly protonated species of only two of these conotoxins, alpha-PnIA and alpha-PnIB, showed additional related ions at +40 m/z (+80 Da), indicating the presence of either sulfation or phosphorylation in both components. High-performance liquid chromatographic (HPLC) fractions containing these two conotoxins were examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in both positive and negative ion modes, as well as by MALDI high-energy collision-induced dissociation. These experiments established the presence of a single sulfated tyrosine residue within both alpha-PnIA and alpha-PnIB. Hence their post-translationally modified sequences are GCCSLPPCAANNPDY(S)C-NH2 (alpha-PnIA) and GCCSLPPCALSNPDY(S)C-NH2 (alpha-PnIB). This assignment was supported by comparison of their mass spectral behavior with that of known sulfated and phosphorylated peptides. This data clarified further the distinguishing features of the ionization and fragmentation of such modified peptides. Selective disulfide folding of synthetic alpha-PnIB demonstrated that both sulfated and non-sulfated toxins co-elute on reversed-phase HPLC and that alpha-PnIB possesses the same disulfide connectivity as other 'classical' alpha-conotoxins reported previously.

Identification of tyrosine sulfation in Conus pennaceus conotoxins α-PnIA and α-PnIB: further investigation of labile sulfo- and phosphopeptides by electrospray, matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry Dedicated to the memory of Professor Dr Wilhelm J. Richter.

Identification of tyrosine sulfation in Conus pennaceus conotoxins α-PnIA and α-PnIB: further investigation of labile sulfo- and phosphopeptides by electrospray, matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry Dedicated to the memory of Professor Dr Wilhelm J. Richter.