Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation.

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components.

Matrix-assisted laser desorption/ionization imaging protocol for in situ characterization of tryptic peptide identity and distribution in formalin-fixed tissue.

Rationale: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry provides the means to map the in situ distribution of tryptic peptides in formalin-fixed clinical tissue samples. The ability to analyze clinical samples is of great importance to further developments in the imaging field. However, there is a requirement in this field of research for additional methods describing the characterization of tryptic peptides by MALDI imaging.

Tryptic peptide reference data sets for MALDI imaging mass spectrometry on formalin-fixed ovarian cancer tissues.

MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis. However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed m/z species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue microarray cores, from 31 ovarian cancer patients, by LC-MS/MS.

Internal calibrants allow high accuracy peptide matching between MALDI imaging MS and LC-MS/MS.

One of the important challenges for MALDI imaging mass spectrometry (MALDI-IMS) is the unambiguous identification of measured analytes. One way to do this is to match tryptic peptide MALDI-IMS m/z values with LC-MS/MS identified m/z values. Matching using current MALDI-TOF/TOF MS instruments is difficult due to the variability of in situ time-of-flight (TOF) m/z measurements.

Proteomic analyses using Grifola frondosa metalloendoprotease Lys-N.

Proteomic analysis typically has been performed using proteins digested with trypsin because of the excellent fragmentation patterns they produce in collision induced dissociation (CID). For analyses in which high protein coverage is desirable, such as global monitoring of post-translational modifications, additional sequences can be seen using parallel digestion with a second enzyme. We have benchmarked a relatively obscure basidomycete-derived zinc metalloendopeptidase, Lys-N, that selectively cleaves the amide bond N-terminal of lysine residues.