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Top-down and bottom-up identification of proteins by liquid extraction surface analysis mass spectrometry of healthy and diseased human liver tissue.

Sarsby J, Martin NJ, Lalor PF, Bunch J, Cooper HJ - J. Am. Soc. Mass Spectrom. (2014)

Bottom Line: The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible.MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein.The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies.

View Article: PubMed Central - PubMed

Affiliation: Physical Sciences of Imaging in the Biomedical Sciences Doctoral Training Centre, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

ABSTRACT
Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15-25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies.

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(a) Total number of proteins identified from healthy human liver tissue using a bottom-up approach following LESA extraction using solvents comprising 50 mM NH4(HCO3), 70% methanol, and 50% methanol. (b) Sequence coverage obtained for FABP1 following 50 mM NH4(HCO3) extraction, automated tryptic digestion, and LC MS/MS from two technical replicates
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Fig1: (a) Total number of proteins identified from healthy human liver tissue using a bottom-up approach following LESA extraction using solvents comprising 50 mM NH4(HCO3), 70% methanol, and 50% methanol. (b) Sequence coverage obtained for FABP1 following 50 mM NH4(HCO3) extraction, automated tryptic digestion, and LC MS/MS from two technical replicates

Mentions: Intact proteins were extracted from healthy human liver tissue via automated direct surface sampling by LESA and subjected to automated trypsin digestion using the method described by Martin et al. [8]. In that work, Martin et al. found that 1 h was the optimum digestion time. Three extraction solvents were considered: 70% (v/v) methanol, 50% (v/v) methanol, and 50 mM ammonium bicarbonate. The three extraction solvents were applied for LESA of three serial sections from a tumor resection margin. For each extraction solvent composition, two technical replicates were performed. Two searches of the data against the protein database were performed in order to determine the effect of the short digestion time. In the first, two missed trypsin cleavages were allowed, and in the second, three missed cleavages were allowed. When two missed cleavages were allowed, a total of 475 non-redundant proteins were detected: 428 proteins were detected following the ammonium bicarbonate extraction; 200 were detected following the 50% methanol extraction, and 99 were detected following the 70% methanol extraction. When three missed cleavages were allowed, 549 non-redundant proteins were detected: 464 proteins were detected following the ammonium bicarbonate extraction; 214 were detected following the 50% methanol extraction, and 116 were detected following the 70% methanol extraction. See Figure 1a. Proteins identified are detailed in Supplementary File 1. Single peptide protein identifications are presented in Supplementary File 2. Increasing the organic solvent content of the extraction solution resulted in fewer identifications, and this is perhaps not surprising. This observation has relevance to the top-down analysis and is discussed further below.Figure 1


Top-down and bottom-up identification of proteins by liquid extraction surface analysis mass spectrometry of healthy and diseased human liver tissue.

Sarsby J, Martin NJ, Lalor PF, Bunch J, Cooper HJ - J. Am. Soc. Mass Spectrom. (2014)

(a) Total number of proteins identified from healthy human liver tissue using a bottom-up approach following LESA extraction using solvents comprising 50 mM NH4(HCO3), 70% methanol, and 50% methanol. (b) Sequence coverage obtained for FABP1 following 50 mM NH4(HCO3) extraction, automated tryptic digestion, and LC MS/MS from two technical replicates
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4197381&req=5

Fig1: (a) Total number of proteins identified from healthy human liver tissue using a bottom-up approach following LESA extraction using solvents comprising 50 mM NH4(HCO3), 70% methanol, and 50% methanol. (b) Sequence coverage obtained for FABP1 following 50 mM NH4(HCO3) extraction, automated tryptic digestion, and LC MS/MS from two technical replicates
Mentions: Intact proteins were extracted from healthy human liver tissue via automated direct surface sampling by LESA and subjected to automated trypsin digestion using the method described by Martin et al. [8]. In that work, Martin et al. found that 1 h was the optimum digestion time. Three extraction solvents were considered: 70% (v/v) methanol, 50% (v/v) methanol, and 50 mM ammonium bicarbonate. The three extraction solvents were applied for LESA of three serial sections from a tumor resection margin. For each extraction solvent composition, two technical replicates were performed. Two searches of the data against the protein database were performed in order to determine the effect of the short digestion time. In the first, two missed trypsin cleavages were allowed, and in the second, three missed cleavages were allowed. When two missed cleavages were allowed, a total of 475 non-redundant proteins were detected: 428 proteins were detected following the ammonium bicarbonate extraction; 200 were detected following the 50% methanol extraction, and 99 were detected following the 70% methanol extraction. When three missed cleavages were allowed, 549 non-redundant proteins were detected: 464 proteins were detected following the ammonium bicarbonate extraction; 214 were detected following the 50% methanol extraction, and 116 were detected following the 70% methanol extraction. See Figure 1a. Proteins identified are detailed in Supplementary File 1. Single peptide protein identifications are presented in Supplementary File 2. Increasing the organic solvent content of the extraction solution resulted in fewer identifications, and this is perhaps not surprising. This observation has relevance to the top-down analysis and is discussed further below.Figure 1

Bottom Line: The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible.MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein.The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies.

View Article: PubMed Central - PubMed

Affiliation: Physical Sciences of Imaging in the Biomedical Sciences Doctoral Training Centre, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

ABSTRACT
Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15-25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies.

Show MeSH
Related in: MedlinePlus