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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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Related in: MedlinePlus

Expanded m/z regions from the full scan mass spectra obtained following LESA MS analysis of two healthy human liver sections (a) and (b), and two NASH human liver sections (c) and (d). Labeled peaks are FABP1 (+16 charge state) (blue circle), FABP1TA (+16 charge state) (green triangle), and α-Hb (+16 charge state) (red square)
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Fig6: Expanded m/z regions from the full scan mass spectra obtained following LESA MS analysis of two healthy human liver sections (a) and (b), and two NASH human liver sections (c) and (d). Labeled peaks are FABP1 (+16 charge state) (blue circle), FABP1TA (+16 charge state) (green triangle), and α-Hb (+16 charge state) (red square)

Mentions: Figure 6 shows expanded m/z regions containing the +16 charge state of FABP1 and FABP1TA of the mass spectra obtained for the four samples analyzed. Figure 6a and b show the mass spectra from healthy (non-diseased) tissue. In one sample, only FABP1 is observed, whereas in the other both FABP1 and the variant are observed. Figure 6c and d show the mass spectra from NASH tissue. Similarly, one sample contains the variant alone, whereas the other reveals the presence of both FABP1 and FABP1TA. As described above, it has been demonstrated recently that individuals with FABPTA have an increased risk of developing NASH [6]. In addition, it was shown that another polymorphism compounds that risk. The observation of the variant in non-diseased tissue could be explained by the risk associated with FABPTA being attenuated by other factors. It is worth noting, however, that the sample containing both FAPB1 and the variant was from a tumor resection margin, whereas the sample containing FAPB1 alone was from a liver rejected for transplant because of the presence of a small lesion. The non-diseased samples are also characterized by reduced signals for α-hemoglobin. In the case of the liver rejected for transplant (Figure 6a), the organ was perfused with buffer (i.e., blood was removed) following removal from the donor. In the case of the tumor resection margin (Figure 6b), it is likely that the surgical procedure resulted in local hemolysis and reduced blood supply.Figure 6


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)

Expanded m/z regions from the full scan mass spectra obtained following LESA MS analysis of two healthy human liver sections (a) and (b), and two NASH human liver sections (c) and (d). Labeled peaks are FABP1 (+16 charge state) (blue circle), FABP1TA (+16 charge state) (green triangle), and α-Hb (+16 charge state) (red square)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: Expanded m/z regions from the full scan mass spectra obtained following LESA MS analysis of two healthy human liver sections (a) and (b), and two NASH human liver sections (c) and (d). Labeled peaks are FABP1 (+16 charge state) (blue circle), FABP1TA (+16 charge state) (green triangle), and α-Hb (+16 charge state) (red square)
Mentions: Figure 6 shows expanded m/z regions containing the +16 charge state of FABP1 and FABP1TA of the mass spectra obtained for the four samples analyzed. Figure 6a and b show the mass spectra from healthy (non-diseased) tissue. In one sample, only FABP1 is observed, whereas in the other both FABP1 and the variant are observed. Figure 6c and d show the mass spectra from NASH tissue. Similarly, one sample contains the variant alone, whereas the other reveals the presence of both FABP1 and FABP1TA. As described above, it has been demonstrated recently that individuals with FABPTA have an increased risk of developing NASH [6]. In addition, it was shown that another polymorphism compounds that risk. The observation of the variant in non-diseased tissue could be explained by the risk associated with FABPTA being attenuated by other factors. It is worth noting, however, that the sample containing both FAPB1 and the variant was from a tumor resection margin, whereas the sample containing FAPB1 alone was from a liver rejected for transplant because of the presence of a small lesion. The non-diseased samples are also characterized by reduced signals for α-hemoglobin. In the case of the liver rejected for transplant (Figure 6a), the organ was perfused with buffer (i.e., blood was removed) following removal from the donor. In the case of the tumor resection margin (Figure 6b), it is likely that the surgical procedure resulted in local hemolysis and reduced blood supply.Figure 6

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