Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma.

We report a proteomic analysis of microdissected material from formalin-fixed and paraffin-embedded colorectal cancer, quantifying > 7500 proteins between patient matched normal mucosa, primary carcinoma, and nodal metastases. Expression levels of 1808 proteins changed significantly between normal and cancer tissues, a much larger fraction than that reported in transcript-based studies. Tumor cells exhibit extensive alterations in the cell-surface and nuclear proteomes.

Mapping N-glycosylation sites across seven evolutionarily distant species reveals a divergent substrate proteome despite a common core machinery.

N-linked glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteomes in nonmammalian systems. Here, we measure N-glycoproteomes of the major model organisms Arabidopsis thaliana, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio, representatively spanning the eukaryotic domain of life. The number of detected N-glycosylation sites varied between 425 in fission yeast, 516 in budding yeast, 1,794 in worm, 2,186 in plant, 2,229 in fly, and 2,254 in zebrafish.

Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method.

The filter-aided sample preparation (FASP) method allows gel-free processing of biological samples solubilized with detergents for proteomic analysis by mass spectrometry. In FASP detergents are removed by ultrafiltration, and after protein digestion peptides are separated from undigested material. Here we compare the effectiveness of different filtration devices for analysis of proteomes and glycoproteomes.

Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria.

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms.

Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.

N-linked glycosylation is a biologically important protein modification, but only a small fraction of modification sites have been mapped. We developed a "filter aided sample preparation" (FASP)-based method in which glycopeptides are enriched by binding to lectins on the top of a filter and mapped 6367 N-glycosylation sites on 2352 proteins in four mouse tissues and blood plasma using high-accuracy mass spectrometry. We found 74% of known mouse N-glycosites and discovered an additional 5753 sites on a diverse range of proteins.

Proteome, phosphoproteome, and N-glycoproteome are quantitatively preserved in formalin-fixed paraffin-embedded tissue and analyzable by high-resolution mass spectrometry.

Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of post-translational modifications is thought to be difficult or impossible. The filter aided sample preparation (FASP) method can analyze proteomic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis.

Suppression of water as a nucleophile in Candida antarctica lipase B catalysis.

A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mM butanol 14 times faster than hydrolysis.

Caenorhabditis elegans has a phosphoproteome atypical for metazoans that is enriched in developmental and sex determination proteins.

In eukaryotic species, signal transduction is often mediated by posttranslational modifications that can serve as regulatory switches. Although nematodes have usually been studied by genetic rather than biochemical methods, PTMs such as phosphorylation are thought to control all aspects of biological functions including sex determination and development. Here, we apply high accuracy mass spectrometry and comprehensive bioinformatic analysis to determine and characterize the in vivo Caenorhabditis elegans phosphoproteome for the first time.