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.
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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)