Concentration-Dependent <i>h</i>MSC Differentiation on Orthogonal Concentration Gradients of GRGDS and BMP‑2 Peptides

Self-assembled monolayer substrates containing tethered orthogonal concentration profiles of GRGDS (glycine/arginine/glycine/aspartic acid/serine) and BMP-2 (bone morphogenetic protein) peptides are shown to accelerate or decelerate, depending on the concentrations, the proliferation and osteoblastic differentiation of human mesenchymal stem cell (<i>h</i>MSC) populations in vitro without the use of osteogenic additives in culture medium. Concurrently, the single peptide gradient controls (GRGDS or BMP-2 only) induce significantly different proliferation and differentiation behavior from the orthogonal substrates. Bone sialoprotein (BSP) and Runt-related transcription factor 2 (Runx2) PCR data acquired from <i>h</i>MSC populations isolated by laser capture microdissection correspond spatially and temporally to protein marker data obtained from immunofluorescent imaging tracking of the differentiation process. Although genomic and protein data at high concentrations area GRGDS (71–83 pmol/cm²):BMP-2 (25 pmol/cm²) reveal an implicit acceleration on the <i>h</i>MSC differentiation timeline relative to the individual peptide concentrations, most of the GRGDS and BMP-2 combinations displayed significant antagonistic behavior during the <i>h</i>MSC differentiation. These data highlight the utility of the orthogonal gradient approach to aid in identifying optimal concentration ranges of translationally relevant peptides and growth factors for targeting cell lineage commitment

Global Post-Translational Modification Discovery

A new global post-translational modification (PTM) discovery strategy, G-PTM-D, is described. A proteomics database containing UniProt-curated PTM information is supplemented with potential new modification types and sites discovered from a first-round search of mass spectrometry data with ultrawide precursor mass tolerance. A second-round search employing the supplemented database conducted with standard narrow mass tolerances yields deep coverage and a rich variety of peptide modifications with high confidence in complex unenriched samples. The G-PTM-D strategy represents a major advance to the previously reported G-PTM strategy and provides a powerful new capability to the proteomics research community