7 research outputs found
Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes
Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production
Lipid droplets are vital to hepatitis C virus (HCV) infection as the putative sites of virion assembly, but morphogenesis and egress of virions remain ill defined. We performed quantitative lipid droplet proteome analysis of HCV-infected cells to identify co-factors of that process. Our results demonstrate that HCV disconnects lipid droplets from their metabolic function. Annexin A3 (ANXA3), a protein enriched in lipid droplet fractions, strongly impacted HCV replication and was characterized further: ANXA3 is recruited to lipid-rich fractions in HCV-infected cells by the viral core and NS5A proteins. ANXA3 knockdown does not affect HCV RNA replication but severely impairs virion production with lower specific infectivity and higher density of secreted virions. ANXA3 is essential for the interaction of viral envelope E2 with apolipoprotein E (ApoE) and for trafficking, but not lipidation, of ApoE in HCV-infected cells. Thus, we identified ANXA3 as a regulator of HCV maturation and egress
Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes
The complexity of
mammalian proteomes is a challenge in bottom-up
proteomics. For a comprehensive proteome analysis, multidimensional
separation strategies are necessary. Online two-dimensional liquid
chromatography–tandem mass spectrometry (2D-LC-MS/MS) combining
strong cation exchange (SCX) in the first dimension with reversed-phase
(RP) chromatography in the second dimension provides a powerful approach
to analyze complex proteomes. Although the combination of SCX with
RP chromatography provides a good orthogonality, only a moderate separation
is achieved in the first dimension for peptides with two (+2) or three
(+3) positive charges. The aim of this study was to improve the performance
of online SCX-RP-MS/MS by applying displacement chromatography to
the first separation dimension. Compared to gradient chromatography
mode (GCM), displacement chromatography mode (DCM) was expected to
improve the separation of +2-peptides and +3-peptides, thus reducing
complexity and increasing ionization and detectability. The results
show that DCM provided a separation of +2-peptides and +3-peptides
in remarkably sharp zones with a low degree of coelution, thus providing
fractions with significantly higher purities compared to GCM. In particular,
+2-peptides were separated over several fractions, which was not possible
to achieve in GCM. The better separation in DCM resulted in a higher
reproducibility and significantly higher identification rates for
both peptides and proteins including a 2.6-fold increase for +2-peptides.
The higher number of identified peptides in DCM resulted in significantly
higher protein sequence coverages and a considerably higher number
of unique peptides per protein. Compared to conventionally used salt-based
GCM, DCM increased the performance of online SCX-RP-MS/MS and enabled
comprehensive proteome profiling in the low microgram range
Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes
Differential regulation of extracellular matrix proteins in three recurrent liver metastases of a single patient with colorectal cancer
Colorectal cancer (CRC) patients suffer from the second highest mortality among all cancer entities. In half of all CRC patients, colorectal cancer liver metastases (CRLM) can be observed. Metastatic colorectal cancer is associated with poor overall survival and limited treatment options. Even after successful surgical resection of the primary tumor, metachronous liver metastases occur in one out of eight cases. The only available curative intended treatment is hepatic resection, but metachronous CRLM frequently recur after approximately 1 year. In this study, we performed a proteome analysis of three recurrent liver metastases of a single CRC patient by mass spectrometry. Despite surgical resection of the primary CRC and adjuvant chemotherapy plus cetuximab treatment, the patient developed three metachronous CRLM which occurred consecutively after 9, 21 and 31 months. We identified a set of 1132 proteins expressed in the three metachronous CRLM, of which 481 were differentially regulated, including 81 proteins that were associated with the extracellular matrix (ECM). 56 ECM associated proteins were identified as upregulated in the third metastasis, 26 (46%) of which were previously described as negative prognostic markers in CRC, including tenascin C, nidogen 1, fibulin 1 and vitronectin. These data may reflect an ascending trend of malignancy from the first to the third metachronous colorectal cancer liver metastasis. Additionally, the results indicate different ECM phenotypes for recurrent metachronous metastasis, associated with different grades of malignancy and highlights the importance of individual analysis of molecular features in different, consecutive metastatic events in a single patient