Limits...
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.

Show MeSH

Related in: MedlinePlus

ETD mass spectra obtained from +16 ions of (a) FABP1 and (b) FABPTA following LESA of NASH human liver sections. Insets: observed sequence coverage: c/z ions are shown in red, a/y ions in black
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4197381&req=5

Fig5: ETD mass spectra obtained from +16 ions of (a) FABP1 and (b) FABPTA following LESA of NASH human liver sections. Insets: observed sequence coverage: c/z ions are shown in red, a/y ions in black

Mentions: Ions of m/z 942.23 and 940.29 (+16 charge state) (corresponding to FABP1 (MWmeas 14111.3943, MWcalc 14111.3892, Δ 2.4 ppm) and FABP1TA (MWmeas 14081.2845, MWcalc 14081.3687, Δ 6.0 ppm) from a NASH sample were subjected to ETD MS/MS. The sequence coverages obtained were 38% and 29%, see Supplementary Tables 7 and 8. The ETD MS/MS spectra of FABP1 and FABP1TA from NASH tissue are shown in Figure 5.Figure 5


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)

ETD mass spectra obtained from +16 ions of (a) FABP1 and (b) FABPTA following LESA of NASH human liver sections. Insets: observed sequence coverage: c/z ions are shown in red, a/y ions in black
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4197381&req=5

Fig5: ETD mass spectra obtained from +16 ions of (a) FABP1 and (b) FABPTA following LESA of NASH human liver sections. Insets: observed sequence coverage: c/z ions are shown in red, a/y ions in black
Mentions: Ions of m/z 942.23 and 940.29 (+16 charge state) (corresponding to FABP1 (MWmeas 14111.3943, MWcalc 14111.3892, Δ 2.4 ppm) and FABP1TA (MWmeas 14081.2845, MWcalc 14081.3687, Δ 6.0 ppm) from a NASH sample were subjected to ETD MS/MS. The sequence coverages obtained were 38% and 29%, see Supplementary Tables 7 and 8. The ETD MS/MS spectra of FABP1 and FABP1TA from NASH tissue are shown in Figure 5.Figure 5

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