Sample preparation techniques for the untargeted LC-MS-based discovery of peptides in complex biological matrices

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.

Figure 1

Illustrative iceberg representation of high dynamic range of proteins found in blood, showing various classes of proteins and peptides (figure composed of literature data [21] and others). Four arbitrary assemblies of proteins/peptides can be made, and representative species are indicated. Iceberg tip contains abundant classical plasma proteins detectable in 1 μL of sample or less. Tissue leakage proteins typically require, at least, 1 mL plasma volumes (typically after depletion of interfering abundant proteins), and concentration ranges of secreted signal peptides/proteins like insulin, somatotropin are yet another 3 orders of magnitude lower. Interleukins and other cytokines really push current MS systems to their very limit, whereas other neurosecretory signal peptides require extensive concentration steps to reach levels detectable by MS.

Figure 2

Schematic overview of methods and techniques used in proteome and peptidome analyses for sample preparation prior to LC MS/MS.

Figure 3

Comparison of the yield of low molecular weight protein/peptide extraction from serum by means of differential solubilization (DS), organic precipitation (OP), and ultrafiltration (UF). All techniques effectively remove the high molecular weight serum protein, whereas recovery of LMW proteins/peptides is highest with DS. Reprinted with permission from Kawashima et al. [94]. Copyright 2011 American Chemical Society.

Figure 4

Effect on the number of detected (neuro)peptides (from mouse hypothalamus) by postmortem time (time between tissue collection and heat denaturation/stabilization) [104]. (a) Number of detected peptides by nano-LC ESI MS rapidly increases with postmortem time. (b, c) Two-dimensional peptide displays of control group and 10 min postmortem group, respectively. Picture adapted from Sköld et al. [104].