Abstract
Although big progress has been made in sample pretreatment over the last years, there are still considerable limitations when it comes to overcoming complexity and dynamic range problems associated with peptide analyses from biological matrices. Being the little brother of proteomics, peptidomics is a relatively new field of research aiming at the direct analysis of the small proteins, called peptides, many of which are not amenable for typical trypsin-based analytics. In this paper, we present an overview of different techniques and methods currently used for reducing a sample's complexity and for concentrating low abundant compounds to enable successful peptidome analysis. We focus on techniques which can be employed prior to liquid chromatography coupled to mass spectrometry for peptide detection and identification and indicate their advantages as well as their shortcomings when it comes to the untargeted analysis of native peptides from complex biological matrices.
Highlights
Peptides are small proteins, built up of amino acids connected by peptide bonds
Big progress has been made in sample pretreatment over the last years, there are still considerable limitations when it comes to overcoming complexity and dynamic range problems associated with peptide analyses from biological matrices
We focus on techniques which can be employed prior to liquid chromatography coupled to mass spectrometry for peptide detection and identification and indicate their advantages as well as their shortcomings when it comes to the untargeted analysis of native peptides from complex biological matrices
Summary
Peptides are small (low molecular weight, LMW) proteins, built up of amino acids connected by peptide bonds. When studying the intracellular proteome (or peptidome), cells are washed several times with FCS-free cell culture medium and/or phosphate-buffered saline (PBS) prior to lysis to reduce contamination of the sample with (bovine) serum proteins. The only exception to this is the comparison of dye-based depletion to immunodepletion of albumin (see above) In this case, immunodepletion invariably resulted in the most efficient enrichment of low abundant proteins and peptides [62, 65, 66]. In those cases where larger members of the peptidome need to be addressed, another crucial part of sample pretreatment is alkylation and digestion of the peptides/small proteins This can be performed prior to or after the previously described techniques, that is, just before LC-MS/MS analysis. A good example is metalloendopeptidase Lys-N, which guarantees a prominent positive charge at the aminoterminus of the cleaved product [80]
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