Proteomics: From relative to absolute quantification for systems biology approaches - Methods in Microbiology - page 81

post-translational modification (PTM) events, for example, changes in MW due to
proteolytic processing/degradation or direct staining of phosphorylation events by
ProQ Diamond ( Hecker et al. , 2008, 2009; Rabilloud et al. , 2009 ). Differential
gel image analysis of 2D gels representing different proteomic snapshots delivers
relative quantitative information solely based on the staining intensities of the pro-
tein spots found on the gels. Since the invention of 2DE, this technique has matured
to become an exceedingly robust and relatively cheap technique in terms of equip-
ment needed ( Westermeier and Marouga, 2005; Rabilloud et al. , 2010 ). In microbi-
ology, 2D gel-based proteomics is commonly used for assessment of adaptation
responses to nutrient shifts ( Bernhardt et al. , 1999 ), antimicrobial agents ( Wenzel
and Bandow, 2011 ) or environmental stresses and starvation ( Budde et al. , 2006 ).
The advantages of 2D gels for the study of microbial physiology, namely, the visu-
alization of the majority of the proteins involved in biosynthetic pathways and the
main metabolic routes, have recently been reviewed for Gram-positive bacteria
( V ¨ lker and Hecker, 2005; Hecker et al. , 2008 ). Elucidating changes in the amounts
of key effectors in metabolism and cellular structure provides valuable insights into
the main processes of life and is thus essential for systems biology approaches.
1.3 Gel-free proteomics
With the advent of high-resolution and accurate mass spectrometers, facilitating the
analysis of complex peptide mixtures by so-called shot-gun proteomics, gel-free
studies have gained exceeding significance in the field ( Cox and Mann, 2007,
2011; Michalski et al. , 2011 ). In contrast to the analysis of intact proteins in classic
(i.e. gel-based) proteomics experiments, gel-free proteomics starts with an enzymatic
digestion of a protein sample. With this step, information on the co-occurrence of
PTMs on particular protein species or proteolytic processing is largely lost. The
resulting complex peptide mix is then subjected to separation by liquid chromato-
graphy and analysis by mass spectrometry. Bioinformatic post-processing of the
acquired data maps the peptides to the masses determined by liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS) and, finally, to the assembly/
grouping of the peptide data to the protein data. Quantitative information is more
difficult to obtain for gel-free proteomics experiments unless stable isotopes are
incorporated in the samples prior to analysis ( Bantscheff et al. , 2007 ). Gel-free pro-
teomics workflows have evolved rapidly in recent years, circumventing gel-based
limitations with respect to MW, p I and/or hydrophobicity, thereby setting new stan-
dards in the sensitivity, versatility and comprehensiveness of proteomics studies
( Cox and Mann, 2007; de Godoy et al. , 2008 ). In microbial proteomics, gel-free tech-
niques have pushed the limits far beyond those associated with studies relying on
classical proteomics techniques with respect to the depth covered in relative quan-
titative studies. In gel-free proteome studies, relative quantitation can be based on a
wide array of techniques ( Bantscheff et al. , 2012 ). However, due to the ease with
which components in growth media can be exchanged, metabolic labelling has set
the standards in microbial gel-free proteomics. In addition to metabolic labelling