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with 15 N and the widely applied SILAC methodology, tagging strategies such as
iTRAQ have proven their applicability in large-scale proteome studies ( Wolff
et al. , 2006; Dreisbach et al. , 2008; Soufi et al. , 2010 ). For Gram-positive bacteria,
such as Staphylococcus aureus , Bacillus subtilis and Corynebacterium glutamicum ,
in-depth proteome studies with almost complete coverage of the main metabolic
pathways, as well as subcellular fractions such as the membrane, surface and secreted
proteomes have provided new insights in their physiology and pathophysiology
( Becher et al. , 2009; Otto et al. , 2010; Becher et al. , 2011; Poetsch et al. , 2011 ).
The main limitation of gel-free proteomics technologies is the requirement for
expensive and resource-intense mass spectrometry instrumentation.
1.4 Targeted proteomics
In recent years, in parallel with discovery-driven experiments aimed at cataloguing
the largest possible number of identified proteins, targeted methods have been devel-
oped for monitoring and quantifying selected proteins of interest ( Picotti and
Aebersold, 2012 ). Quantitative, discovery-based mass spectrometric experiments
require, in general, the identification of the same subset of proteins in each sample
to be compared. Relative quantitative information is then derived from the signal
intensities of the ion masses of the peptides that were assigned to the target proteins.
Despite the advantage of a large number of identified proteins in every sample, this
leads to an inevitable excessive redundancy in identification data.
Limitations in quantification have stimulated the development of alternative
approaches that rely on existing peptide identification data and fragmentation patterns
in target-oriented proteomics experiments ( Schmidt et al. , 2009; Domon and
Aebersold, 2010 ). In selected reaction monitoring (SRM)-based targeted proteomics,
distinct pairs of precursor ion masses and cognate fragmentation masses, derived from
existing datasets or large-scale spectral libraries, are used for sensitive and specific
determination of these pairs (transitions) in triple quadrupole mass spectrometers
(QQQ). QQQ display comparably low resolution and mass accuracy compared to
the high-end mass spectrometric equipment that is used in traditional discovery-based
experiments. However, QQQ instrumentation is outstandingwith respect to its dynamic
range (three to four orders of magnitude of linear response; Kirkpatrick et al. ,2005 )and
speed of acquisition (high duty cycle), leading to good ion statistics and sensitivity.
Consequently, large numbers of samples may be interrogated for relative abundance
of a predetermined number of peptides without the need for time-consuming discov-
ery-based proteomics experiments. Taken together, these features make targeted mass
spectrometry the preferred choice for systems biology approaches ( Lange et al. ,2008 ).
1.5 Proteomics based on data-independent acquisition
As a consequence of the need to generate ever more comprehensive proteomics data-
sets for systems biology approaches and recent developments in mass spectrometric
instrumentation, new workflows based on data-independent acquisition have
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