abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes

Affinity purification coupled to 1-D gel-free liquid chromatography mass spectrometry (LC–MS) is a well-established and widespread approach for the analyses of noncovalently interacting protein complexes. In this study, two proteins conjugated to a streptavidin-binding peptide and hemagglutinin double tag were expressed in the respective Flp-In HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed that the expression level of SH-GFP was ∼50% lower than that of SH-TBK1_MOUSE. Subsequently, the input material was normalized to obtain a similar quantity of purified SH-tagged proteins. Optimization of the release of protein complexes from the <i>anti</i>-HA-agarose with different eluting agents was then assessed. With respect to the total number of protein groups identified in the purified complexes, elution with 2% SDS surpassed both 100 mM glycine and 100 mM formic acid. Relative quantitation of the purified protein complexes using TMT 6-plex reagents confirmed the higher efficiency of the 2% SDS elution followed by filter-aided sample preparation (FASP). The data presented in this study provide a new application of FASP to quantitative MS analysis of affinity-purified protein complexes. We have termed the approach abFASP-MS, or affinity-based filter-aided sample preparation mass spectrometry

abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes

Affinity purification coupled to 1-D gel-free liquid chromatography mass spectrometry (LC–MS) is a well-established and widespread approach for the analyses of noncovalently interacting protein complexes. In this study, two proteins conjugated to a streptavidin-binding peptide and hemagglutinin double tag were expressed in the respective Flp-In HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed that the expression level of SH-GFP was ∼50% lower than that of SH-TBK1_MOUSE. Subsequently, the input material was normalized to obtain a similar quantity of purified SH-tagged proteins. Optimization of the release of protein complexes from the <i>anti</i>-HA-agarose with different eluting agents was then assessed. With respect to the total number of protein groups identified in the purified complexes, elution with 2% SDS surpassed both 100 mM glycine and 100 mM formic acid. Relative quantitation of the purified protein complexes using TMT 6-plex reagents confirmed the higher efficiency of the 2% SDS elution followed by filter-aided sample preparation (FASP). The data presented in this study provide a new application of FASP to quantitative MS analysis of affinity-purified protein complexes. We have termed the approach abFASP-MS, or affinity-based filter-aided sample preparation mass spectrometry

abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes

Affinity purification coupled to 1-D gel-free liquid chromatography mass spectrometry (LC–MS) is a well-established and widespread approach for the analyses of noncovalently interacting protein complexes. In this study, two proteins conjugated to a streptavidin-binding peptide and hemagglutinin double tag were expressed in the respective Flp-In HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed that the expression level of SH-GFP was ∼50% lower than that of SH-TBK1_MOUSE. Subsequently, the input material was normalized to obtain a similar quantity of purified SH-tagged proteins. Optimization of the release of protein complexes from the <i>anti</i>-HA-agarose with different eluting agents was then assessed. With respect to the total number of protein groups identified in the purified complexes, elution with 2% SDS surpassed both 100 mM glycine and 100 mM formic acid. Relative quantitation of the purified protein complexes using TMT 6-plex reagents confirmed the higher efficiency of the 2% SDS elution followed by filter-aided sample preparation (FASP). The data presented in this study provide a new application of FASP to quantitative MS analysis of affinity-purified protein complexes. We have termed the approach abFASP-MS, or affinity-based filter-aided sample preparation mass spectrometry

A Pilot Proteogenomic Study with Data Integration Identifies MCT1 and GLUT1 as Prognostic Markers in Lung Adenocarcinoma

<div><p>We performed a pilot proteogenomic study to compare lung adenocarcinoma to lung squamous cell carcinoma using quantitative proteomics (6-plex TMT) combined with a customized Affymetrix GeneChip. Using MaxQuant software, we identified 51,001 unique peptides that mapped to 7,241 unique proteins and from these identified 6,373 genes with matching protein expression for further analysis. We found a minor correlation between gene expression and protein expression; both datasets were able to independently recapitulate known differences between the adenocarcinoma and squamous cell carcinoma subtypes. We found 565 proteins and 629 genes to be differentially expressed between adenocarcinoma and squamous cell carcinoma, with 113 of these consistently differentially expressed at both the gene and protein levels. We then compared our results to published adenocarcinoma versus squamous cell carcinoma proteomic data that we also processed with MaxQuant. We selected two proteins consistently overexpressed in squamous cell carcinoma in all studies, MCT1 (SLC16A1) and GLUT1 (SLC2A1), for further investigation. We found differential expression of these same proteins at the gene level in our study as well as in other public gene expression datasets. These findings combined with survival analysis of public datasets suggest that MCT1 and GLUT1 may be potential prognostic markers in adenocarcinoma and druggable targets in squamous cell carcinoma. Data are available via ProteomeXchange with identifier PXD002622.</p></div

Kvalitet inom universitets och högskolebibliotek - redovisning av enkät 2008

Ett övergripande problem med utvärdering av informations/biblioteksverksamhet är att det fortfarande nästan enbart är traditionella uppgifter som utvärderas. En annan aspekt är att mäta kvalitet och nytta i andra än ekonomiska termer – d.v.s. kvalitativa indikatorer och mått. Denna rapport innehåller en inventering (ej bedömning) vilka metoder, ansatser och initiativ som idag finns eller håller på att utvecklas på lärosätena/biblioteken för att mäta kvalitet på icke-kvantitativa sätt

A Miniaturized Chemical Proteomic Approach for Target Profiling of Clinical Kinase Inhibitors in Tumor Biopsies

While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 μg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care

Gene and Protein Expression Agreement.

<p>Gene and Protein Expression Agreement.</p

Gene and Protein Expression Show Minor Correlations Across Samples.

<p>Mean intensities are given in log₂ scale. (A) Pairwise protein expression and corresponding gene expression across all samples. (B) Pearson correlations grouped by protein and corresponding gene. Values with Pearson R > 0 are colored yellow. The mean Pearson correlation, including all positive and negative correlations, was 0.34.</p

Comparison with Existing NSCLC Proteomic Datasets.

<p>Mean intensities are given in log₂ scale. (A) The correlations between reporter ion intensities and peptide intensities from Kikuchi et al. were low (R = 0.3, <i>P</i> < 2.2E-16; ρ = 0.26, <i>P</i> < 2.2E-16). (B) As with the Kikuchi et al. data, correlations between reporter ion intensities and peptide intensities from Li et al. were also low (R = 0.23, <i>P</i> < 2.2E-16; ρ = 0.21, <i>P</i> < 2.2E-16).</p

A Miniaturized Chemical Proteomic Approach for Target Profiling of Clinical Kinase Inhibitors in Tumor Biopsies

While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 μg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care