Abstract
Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual’s fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis.Graphical ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1140-z) contains supplementary material, which is available to authorized users.
Highlights
I n Matrix Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI), the identification of observed proteins remains a challenge primarily due to the drop in sensitivity of time of flight (TOF) mass spectrometers beyond the mass range 25–30 kDa [1], inadequate mass resolving power at those molecular weights, as well as limited capabilities for top down approaches applied to singly charged ions and within samples with more than one protein
In Situ Fingermark Digestion for MALDI MSP Trypsin solution (20 μg/mL reconstituted in 50 mM ammonium bicarbonate, pH 8) containing either 10 mM OcGlu, Octyl 1-thio-ß-Dglucopyranoside (OcThio), Decyl ß-D-maltoside (DDM), MEGA8 or RapiGest SF, in concentrations varying from 0.5% to 2% w/v, was manually deposited (0.5 μL droplets) onto the fingermarks
This study aimed to investigate the efficiency of a range of potentially useful detergents to increase the ion abundance and ion population from fingermark protein-originating peptides, within a shotgun proteomic approach involving in situ proteolysis prior to MALDI MS analysis
Summary
I n Matrix Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI), the identification of observed proteins remains a challenge primarily due to the drop in sensitivity of time of flight (TOF) mass spectrometers beyond the mass range 25–30 kDa [1], inadequate mass resolving power at those molecular weights, as well as limited capabilities for top down approaches applied to singly charged ions and within samples with more than one protein. (purified protein samples or from tissue homogenates), methodologies have been devised to digest proteins in situ; these protocols are applied to understand the function-localization relationship through preserving protein localization within a tissue Though very informative, this strategy typically appears to yield a small number of identifiable peptides of low ion intensity when analyzed by mass spectrometry (MS); usually 20 at the most are identified by direct measurements [2]. This strategy typically appears to yield a small number of identifiable peptides of low ion intensity when analyzed by mass spectrometry (MS); usually 20 at the most are identified by direct measurements [2] This is very poor in comparison to conventional proteomics methodology (i.e., LC/ESI MSMS) applied to in-solution digests where several thousand peptides might be expected to be identified. An important step in obtaining suitable and reliable protein signatures
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Journal of the American Society for Mass Spectrometry
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
