Compatible with previous in vitro observations, in vivo-acquired DAP(R) in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.

Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed.

Acquisition of tandem mass spectra from peptides or other analytes deposited on non-conductive membranes is inhibited on instruments combining matrix-assisted laser desorption/ionization with tandem time-of-flight analyzers (MALDI-TOF/TOF) due to a charging effect. A thin layer of gold renders the membrane conductive. This allows adequate data acquisition on MALDI-TOF/TOF systems. Therefore, this methodology extends the capacity of the molecular scanner concept to tandem mass spectrometry.

Workflows in urinary proteomics studies are often complex and require many steps to enrich, purify, deplete and separate the complex mixture. Many of these methods are laborious, timeconsuming and have the potential for error. Although individual steps of these methods have been previously studied, their downstream compatibilities with fractionation technologies such as OffGel electrophoresis have not been investigated. We developed a One-Step sample preparation workflow that simultaneously i) concentrates proteins, ii) purifies by removing salts and other low molecular weight compounds and iii) depletes (albumin) from urine samples. This simple and robust workflow can be multiplexed and is compatible with a diverse range of downstream multidimensional separation technologies. Additionally, due to its high reproducibility and flexibility in processing samples with different volumes and concentrations, it has the potential to be used for standardization of urinary proteomics studies, as well as for studying other body fluids of similar complexity.

In this study, we performed an in-depth characterization of the male pediatric infant urinary proteome by parallel proteomic analysis of normal healthy adult (n =6) and infant (n =6) males and comparison to available published data. A total of 1584 protein groups were identified. Of these, 708 proteins were identified in samples from both cohorts. Although present in both cohorts, 136 of these common proteins were significantly enriched in urine from adults and 94 proteins were significantly enriched in urine from infants. Using Gene Ontology, we found that the infant-enriched or specific subproteome (743 proteins) had an overrepresentation of proteins that are involved in translation and transcription, cellular growth and metabolic processes. In contrast, the adult enriched or specific subproteome (364 proteins) showed an overexpression of proteins involved in immune response and cell adhesion. This study demonstrates that the non-diseased male urinary proteome is quantitatively affected by age, has age-specific subproteomes, and identifies a common subproteome with no age-dependent abundance variations. These findings highlight the importance of age-matching in urinary proteomics.