Alternative Surfactants for Improved Efficiency of In Situ Tryptic Proteolysis of Fingermarks

Journal of The American Society for Mass Spectrometry, Apr 2015

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. Open image in new window Graphical Abstract ᅟ

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Alternative Surfactants for Improved Efficiency of In Situ Tryptic Proteolysis of Fingermarks

Alternative Surfactants for Improved Efficiency of In Situ Tryptic Proteolysis of Fingermarks Ekta Patel 1 Malcolm R. Clench 1 Andy West 0 Peter S. Marshall 0 Nathan Marshall 1 Simona Francese 1 0 GlaxoSmithKline , Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY , UK 1 Biomolecular Research Centre, Sheffield Hallam University , Sheffield, S1 1WB , UK 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. - I trometry Imaging (MALDI-MSI), the identification of obn Matrix Assisted Laser Desorption Ionization Mass Specserved proteins remains a challenge primarily due to the drop in sensitivity of time of flight (TOF) mass spectrometers beyond the mass range 2530 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. To counteract this, a Bbottom up^ approach is often employed; proteolysis yields smaller peptide fragments, typically between 500 and 3000 Da, which are easier to detect and with high mass accuracy. Whilst conventionally enzymatic digestion is carried out in solution (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 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. This small number of identifiable peptides is in part due to the higher complexity and lower Bextraction^ yield of the peptide mixture obtained through the in situ digestions. The integration of ion mobility separation (IMS) within mass spectrometry analysis has shown to mitigate the complexity of the peptide mixture by resolving isobaric species, thus increasing and improving specificity and identification, respectively [3 5]. However, an important step in obtaining suitable and reliable protein signatures (including those from the less abundant proteins, which, in biomarker discovery and pathology diagnostics would have a game-changing effect), lies in improving the efficiency of in situ proteolysis. The literature shows different ways for depositing the endopeptidase trypsin (the most efficient enzyme on tissue) for in situ proteolysis; sprayers produce a homogenous trypsin coating, which is necessary for successful imaging experiments of peptides and for their identification within their original locations [6, 7], whereas other methods, including robotic spotters [4, 8], have been reported to yield higher peptide signal intensity and a more abundant ion population. The advantages and disadvantages of these and other methods of application have been discussed in a recent review [9]. MS compatible detergents have been used to enhance in gel [10] and in solution [11] digestion of hydrophobic proteins, such as membrane proteins. Hydrophobic proteins can be proteolytically resistant to digestion because of inaccessible cleavage sites; therefore, in such instances, the limited number of peptides produced can ultimately affect protein identification. The amphiphilic nature of a detergent improves solubilization by unfol (...truncated)


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Ekta Patel, Malcolm R. Clench, Andy West, Peter S. Marshall, Nathan Marshall, Simona Francese. Alternative Surfactants for Improved Efficiency of In Situ Tryptic Proteolysis of Fingermarks, Journal of The American Society for Mass Spectrometry, 2015, pp. 862-872, Volume 26, Issue 6, DOI: 10.1007/s13361-015-1140-z