Author:

Abstract:

use of peptide display strategy on short-time extracted neurosecretory tissue Since more than a decade, we have been involved in direct tissue (neuro)peptide profiling (and recently also imaging). As one major objective of our studies is to investigate the peptide signaling from tissue, we aim to avoid sample losses and modifications during complex sample preparation procedures as much as possible. This is achieved by the immediate MS analysis of (1) appropriately sized freshly dissected biological tissue, which is air-dried onto a MALDI target after being covered with 1-2 ul of matrix solution, or (2) 10 um cryotome sections of snap frozen tissue. With the improvement of the performance of the MALDI MS(MS) systems employed the peptidome profiles/images visualized have become more and more complex over the years. To explore to what extent direct tissue MALDI analyses suffer from ion suppression effects, we looked into more elaborate peptidome profiling procedures, albeit still with very little tissue dissection time. For this the tissue under investigation (the insect master neuroendocrine gland) was extracted for no longer than 1 min in 10 ul of 50% methanol, 0.1% formic acid. Separated from the tissue of origin, the extract was found to be very stable over time. We have used automated nanospray ESI on a quadrupole TOF instrument (Advion Nanomate on ABI QSTAR), capillary LC on-line with a similar MS/MS system (Waters capLC-QTof combination) and off-line LC MALDI TOF(TOF) (ABI Voyager DESTR and 4700 Proteome Analyzer), for peptide sequencing, profiling and display. A mere 10 ul extract of one neurosecretory gland of a single insect (American cockroach, Periplaneta Americana; African migratory locust, Locusta migratoria; house cricket, Acheta domesticus) yielded material for more than 4 on-line LC MS/MS runs with up to 300 MS signals in the peptide m/z range. The spectra obtained qualify as biomarker for the respective insect species. Automated data dependent LC MS/MS analysis was performed and fragmented peptides were excluded from MS/MS analysis in subsequent LC MS(/MS) runs. Off-line LC-MALDI Peptide Display on the equivalent of 10 insect glands yielded way over 1000 peptide signals. It shall be clear that only a very limited fraction of the observed peptides could successfully be sequenced automatically. However, the fragmentation spectra from Q-TOF and TOF-TOF combinations not seldom are of sufficiently high quality to allow manual sequence interpretation. In selected cases peptides of interest are specifically analyzed in the Nanomate-QSTAR set-up. We are now in the process of identifying as many naturally occurring peptides as possible. The development of a new generation peptide fragmentation interpretation software (not focused on enzymatically cleaved peptides) is high on our priority list.