The former approach is also more compatible with higher acetonitrile concentrations and optimization of the separations between dimensions is possible. According to previous observations, the off-line approach increases the number of detected peptides with MALDI-TOF-MS and electrospray ionization mass spectrometry (ESI-MS), unlike the on-line analysis, which reduces sample manipulation ( Gilar et al., 2005). Gas chromatography (GC) is also applied for sample separation, although less frequently.
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The resulting peptides are then fractionated using the same separation methods including 1D, 2D (strong cation exchange (SCX)/reversed phase (RP)) or 3D (SCX/avidin/RP) LC and this in an online or offline setup. However, complete protein mixtures can also undergo a digest before they are separated. Subsequently, the proteins undergo a digest and the resulting peptide samples are offered to the ion source, in which MALDI and electrospray ionization (ESI) are the most common methods used (discussed in ‘MALDI-TOF/TOF-MS’ and ‘ESI-FT-ICR-MS’, below). 2D-PAGE can detect isoforms, but favors only abundant proteins, shows difficulties in resolving and visualizing proteins with extreme properties (molecular weight and isoelectric point (pI)), has a limited dynamic range, and decreases the detection sensitivity in the mass spectrometer ( Aerts, 2010 Delahunty and Yates, 2005). For several years, 2D-PAGE was the main tool in proteomics to separate proteins, but it has many drawbacks.
vitripennis ( Ye et al., 2010).īefore complex protein mixtures can enter the mass spectrometer, they are separated using SDS-PAGE, liquid chromatography (LC) or two dimensional-poly acrylamide gel electrophoresis (2D-PAGE) in order to reduce the sample complexity ( Steen and Mann, 2004). The combination of Edman degradation and matrix assisted laser desorption ionization-time of flight-MS (MALDI-TOF-MS) was also used to characterize a defensin-like antimicrobial peptide in the venom of N. rubecula (Asgari et al., 2003a), the solitary bee Osmia rufa ( Stöcklin et al., 2010), and the jack jumper ant Myrmecia pilosula ( Davies et al., 2004). This methodology was used for the study of the venoms from the endoparasitoid wasp C. Substantial amounts of hymenopteran venoms have already been analyzed using these MS strategies, sometimes in combination with Edman sequencing, in order to characterize a few venom components. Although the former method is still being used, mass spectrometric analyses have gained more and more interest because of their superior sensitivity and high sample throughput.
However, Edman degradation has the limitation that sequencing is impossible with a blocked amino-terminus due to modifications, and large quantities of purified peptides are needed in comparison with MS ( Favreau et al., 2006). This sequencing method can be carried out on peptides consisting of 70 residues, while mass spectrometric (MS) methods can only sequence peptides up to 25 residues. Single purified venom components can be characterized using Edman degradation whereby peptides or proteins are sequenced by stepwise chemical degradation from the N-terminus, with subsequent identification of the released amino acid derivatives by UV-absorbance spectroscopy. de Graaf, in Parasitoid Viruses, 2012 Tools for Venom Characterizationįor many years, research towards snake and arthropod venoms has been performed with several proteomic tools, to reveal the structure and biological role of these venomous components.
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