Elastase Digests

Abstract

Despite many advances in membrane proteomics during the last decade the fundamental problem of accessing the transmembrane regions itself has only been addressed to some extent. The present study establishes a method for the nano-LC-based analysis of complex membrane proteomes on the basis of a methanolic porcine pancreatic elastase digest to increase transmembrane coverage. Halobacterium salinarium purple and Corynebacterium glutamicum membranes were successfully analyzed by using the new protocol. We demonstrated that elastase digests yield a large proportion of transmembrane peptides, facilitating membrane protein identification. The potential for characterization of a membrane protein through full sequence coverage using elastase is there but is restricted to the higher abundance protein components. Compatibility of the work flow with the two most common mass spectrometric ionization techniques, ESI and MALDI, was shown. Currently better results are obtained using ESI mainly because of the low response of MALDI for strictly neutral peptides. New findings concerning elastase specificity in complex protein mixtures reveal a new prospect beyond the application in shotgun experiments. Furthermore peptide mass fingerprinting with less specific enzymes might be done in the near future but requires an adaptation of current search algorithms to the new proteases.

Expanding Proteome Coverage with CHarge Ordered Parallel Ion aNalysis (CHOPIN) Combined with Broad Specificity Proteolysis

“…55 While unspecific proteases such as Proteinase K have been used in the past on membrane proteins 56 and to analyze interpeptide cross-links, 57 the data analysis still represents a major challenge as cleavage specificity significantly reduces the computational effort for peptide identification. Recent sequence tag 58 or de-novo-based 46,59,60 methods for peptide identification can benefit from the detection of sequence information prior to the application of precursor mass and cleavage specificity to reduce the search space and achieve similar result characteristics as standard search algorithms.…”

Analysis of mouse liver membrane proteins using multidimensional separations and tandem mass spectrometry

“…This is the main cause for the loss of membrane proteins during the experimental procedures. To circumvent the problems associated with the insolubility of the membrane proteins, a "shotgun" digestion approach has been proposed and employed in many analyses [13][14][15][16]. With this approach, a separation system no longer deals with the separation of the intact proteins, but focuses on the separation of the digested peptides.…”

Unbiased Mitoproteome Analyses Confirm Non-canonical RNA, Expanded Codon Translations

“…Natural proteolysis interferes with proteomic analyses based on artificial additions of digestion enzymes [102], [103], [104]. Analyses accounting for natural proteolysis can complete proteome descriptions [105], [106], [107], [108], [109], [110], [111]. Hence unsupervised analyses assuming random cleavage might detect some actual non-tryptic peptides produced by natural proteolysis or spontaneous protein degradation (especially during sample preparation), potentially completing descriptions of non-canonical mitochondrial peptidomes.…”