SUMMARY To provide a detailed analysis of the molecular components and underlying mechanisms associated with ovarian cancer, we performed a comprehensive mass spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by The Cancer Genome Atlas (TCGA), of which 169 were high-grade serous carcinomas (HGSC). Integrating our proteomic measurements with the genomic data yielded a number of insights into disease such as how different copy number alternations influence the proteome, the proteins associated with chromosomal instability, the sets of signaling pathways that diverse genome rearrangements converge on, as well as the ones most associated with short overall survival. Specific protein acetylations associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational modification levels and clinical outcomes in HGSC.

Comprehensive characterization of protein glycosylation is critical for understanding the structure and function of glycoproteins. However, due to the complexity and heterogeneity of glycoprotein conformations, current glycoprotein analyses focus mainly on either the de-glycosylated glycosylation site (glycosite)-containing peptides or the released glycans. Here, we describe a chemoenzymatic method called solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for the comprehensive characterization of glycoproteins that is able to determine glycan heterogeneity for individual glycosites in addition to providing information about the total N-linked glycan, glycosite-containing peptide and glycoprotein content of complex samples. The NGAG method can also be applied to quantitatively detect glycoprotein alterations in total and site-specific glycan occupancies.

Background After myocardial infarction (MI), the left ventricle (LV) undergoes a wound healing response that includes the robust infiltration of neutrophils and macrophages to facilitate removal of dead myocytes as well as turnover of the extracellular matrix (ECM). Matrix metalloproteinase (MMP)-9 is a key enzyme that regulates post-MI LV remodeling. Methods and Results Infarct regions from wild type and MMP-9 null mice (n=8/group) analyzed by glycoproteomics showed that of 541 N-glycosylated proteins quantified, 45 proteins were at least two-fold up- or down-regulated with MMP-9 deletion (all p<0.05). Cartilage intermediate layer protein (CILP) and platelet glycoprotein 4 (CD36) were identified as having the highest fold increase in MMP-9 null mice. By immunoblotting, CD36 but not CILP decreased steadily over the time course post-MI, which identified CD36 as a candidate MMP-9 substrate. MMP-9 was confirmed in vitro and in vivo to proteolytically degrade CD36. In vitro stimulation of day 7 post-MI macrophages with MMP-9 or a CD36 blocking peptide reduced phagocytic capacity. Dual immunofluorescence revealed concomitant accumulation of apoptotic neutrophils in the MMP-9 null group compared to WT. In vitro stimulation of isolated neutrophils with MMP-9 decreased neutrophil apoptosis, indicated by reduced caspase-9 expression. Conclusions Our data reveals a new cell signaling role for MMP-9 through CD36 degradation to regulate macrophage phagocytosis and neutrophil apoptosis.

The analysis of sialylated glycans is critical for understanding the role of sialic acid in normal biological processes as well as in disease. However, the labile nature of sialic acid typically renders routine analysis of this monosaccharide by mass spectrometric methods has been difficult. To overcome this difficulty we pursued derivatization methodologies, extending established acetohydrazide approaches to aniline-based methods, and finally to optimized p-toluidine derivatization. This new quantitative glycoform profiling method using MALDI-TOF in positive ion mode was validated by first comparing N-glycans isolated from fetuin and serum and was then exploited to analyze the effects of increased metabolic flux through the sialic acid pathway in SW1990 pancreatic cancer cells by using a co-labeling strategy with light and heavy toluidine. The latter results established that metabolic flux, in a complementary manner to the more well-known impact of sialyltransferase expression, can critically modulate the sialylation of specific glycans while leaving others virtually unchanged.