Liquid Samples Desorption Electrospray Ionization Mass Spectrometry In Proteomics Applications

A fast and novel strategy for efficient ionization of phosphopeptides in mixturesis introduced in this dissertation, in which the sample is acidified to low pH tosuppress the deprotonation of phosphate groups and then followed by directanalysis by liquid sample desorption electrospray ionization massspectrometry (DESI-MS). This approach direct ionization of analytes with littleor no sample preparation.Phosphorylation is one of the most common post-translational modifications(PTMs) of proteins and it plays an important role in biological processes suchas signal transduction. A major challenge in phosphoprotein analysis by massspectrometry (MS) is the severe suppression of phosphopeptide signal bynon-phosphorylated peptides when phosphoprotein enzymatic digests areionized by electrospray ionization (ESI) in the positive ion mode. Thisdissertation presents a strategy for tackling this problem by using desorptionelectrospray ionization (DESI) after acidifying the sample with hydrochloricacid. Presumably, the addition of HCl suppresses the deprotonation of peptidephosphate groups in solution, thus increasing their ionization efficiency. UnlikeESI which is not compatible with the use of strong acids for possible instrumentcorrosion concern, DESI is tolerant to low pH samples. Casein and ovalbuminprotein digests were chosen as test samples for this concept. Indeed, forcasein and ovalbumin trypsin and Glu-C digests, all of phosphopeptides,including those completely suppressed during electro sonic spray ionization(ESSI, a variant form of ESI) such as the multiply phosphorylated peptideEMEAEpSIpSpSpSEEIVPNpSVEQK, can be clearly detected by DESI-MS. Inaddition, in the case of-casein digest, the intensity of+2ion of the multiplyphosphorylated peptide RELEELNVPGEIVEpSLpSpSpSEESITR was promoted;furthermore, the corresponding+3ion was generated which is of great value in providing increased sequence coverage upon electron-capture dissociation(ECD). This novel method is highly sensitive (e.g., ca.5pmol of casein wasrequired for typical MS spectra acquisition) and rapid as no chromatographicseparation is in need prior to ionization. The methodology is also applicable tothe analysis of sulfopeptides and would have important applications inphosphoproteomics.