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Differential expression of ribosomal proteins in a human metastasis model identified by coupling 2-D liquid chromatography and mass spectrometry.

ABSTRACT: Proteomic profiling of an experimental tumor metastasis model has the potential to identify gene products that can influence this fatal phenotype of tumor cells. In this study, we focused on the notoriously difficult to assess ribosomal protein component of a pair of cell lines which originate from the same tumor but have opposite metastatic capabilities.Cell lysate proteins were separated using a two-dimensional liquid chromatographic system directly coupled to an ESI-TOF mass spectrometer for accurate intact protein MW analysis. Characterization of distinct post-translational modifications and sequence variation within several ribosomal proteins was obtained using monolithic capillary LC/MS/MS, MALDI-MS and -MS/MS.The combination of these techniques enabled the identification of 45 unique ribosomal proteins, several of which were differentially expressed in metastatic M4A4 cells.The described proteomic profiling approach enables the identification of phenotype-associated ribosomal proteins for subsequent functional analyses and disease biomarker development.

SUBMITTER: Kreunin P

PROVIDER: S-EPMC3426217 | biostudies-literature | 2007 Sep-Oct

REPOSITORIES: biostudies-literature

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Differential expression of ribosomal proteins in a human metastasis model identified by coupling 2-D liquid chromatography and mass spectrometry.

Kreunin Paweena P Yoo Chul C Urquidi Virginia V Lubman David M DM Goodison Steve S

Cancer genomics & proteomics 20070901 5

<h4>Background</h4>Proteomic profiling of an experimental tumor metastasis model has the potential to identify gene products that can influence this fatal phenotype of tumor cells. In this study, we focused on the notoriously difficult to assess ribosomal protein component of a pair of cell lines which originate from the same tumor but have opposite metastatic capabilities.<h4>Materials and methods</h4>Cell lysate proteins were separated using a two-dimensional liquid chromatographic system dire ...[more]

PMID: 17993717

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Project description:Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a "discovery" assay, the latter as a "monitoring" assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique ("Hi3" method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.

Dataset Information

Differential expression of ribosomal proteins in a human metastasis model identified by coupling 2-D liquid chromatography and mass spectrometry.

Publications

Differential expression of ribosomal proteins in a human metastasis model identified by coupling 2-D liquid chromatography and mass spectrometry.

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