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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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CID mass spectra of +3 ions of tryptic peptides [TVVQLEGDNKLVTTFK] and [TVVQLEGDNKLVTAFK] from FABP1 and FABP1TA identified following LESA extraction with 50 mM NH4(HCO3) extraction followed by trypsin digestion and LC-MS/MS
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Fig2: CID mass spectra of +3 ions of tryptic peptides [TVVQLEGDNKLVTTFK] and [TVVQLEGDNKLVTAFK] from FABP1 and FABP1TA identified following LESA extraction with 50 mM NH4(HCO3) extraction followed by trypsin digestion and LC-MS/MS

Mentions: FABP1 was detected in each of the six experiments with sequence coverage ranging between 53% and 57% (see Supplementary Figure 1). Figure 1b shows the sequence coverages obtained for the two replicates obtained following extraction with ammonium bicarbonate solution. As described above, FABP1 and its variant implicated in NASH differ by a single amino acid residue (i.e., Thr→Ala at position 94). The tryptic peptide containing residue 94, that is [91-96], was not observed in any of the analyses. (It is possible that an SRM approach may identify the relevant peptide more reproducibly; however, the Orbitrap instrument used in this work is not suited to such an approach). One of the analyses following extraction with ammonium bicarbonate resulted in the identification of the missed-cleavage tryptic peptide [81-96] (m/zmeas 1791.9931, m/zcalc 1791.9851, Δ 4.9 ppm) from FABP1. The CID mass spectrum obtained from the [M+3H]3+ peptide ions is shown in Figure 2a. The fragmentation sequence coverage for the peptide is poor with no cleavage around the potential substitution site. Clearly, as the variant is the result of a single amino acid substitution and the sequences of FAPB1 and its variant are virtually identical, with the exception of peptide [81-96], the peptides assigned to FABP1 in the database search could also have originated from the variant. The human SwissProt database against which the database was searched was supplemented with the FABP1 variant (hereafter referred to as FABP1TA). The peptide [90-96] from FABP1TA was not identified in any of the analyses; however, the missed-cleavage peptide [81-96] was identified in one of the ammonium bicarbonate extraction replicates (m/zmeas 1761.9831, m/zcalc 1761.9745, Δ 4.5 ppm). The CID spectrum is shown in Figure 2b. Again, fragmentation sequence coverage is poor and no fragmentation was observed adjacent to the site of substitution. It should be noted that top-down analysis of this tissue sample (see below) revealed the presence of the variant.Figure 2


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)

CID mass spectra of +3 ions of tryptic peptides [TVVQLEGDNKLVTTFK] and [TVVQLEGDNKLVTAFK] from FABP1 and FABP1TA identified following LESA extraction with 50 mM NH4(HCO3) extraction followed by trypsin digestion and LC-MS/MS
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: CID mass spectra of +3 ions of tryptic peptides [TVVQLEGDNKLVTTFK] and [TVVQLEGDNKLVTAFK] from FABP1 and FABP1TA identified following LESA extraction with 50 mM NH4(HCO3) extraction followed by trypsin digestion and LC-MS/MS
Mentions: FABP1 was detected in each of the six experiments with sequence coverage ranging between 53% and 57% (see Supplementary Figure 1). Figure 1b shows the sequence coverages obtained for the two replicates obtained following extraction with ammonium bicarbonate solution. As described above, FABP1 and its variant implicated in NASH differ by a single amino acid residue (i.e., Thr→Ala at position 94). The tryptic peptide containing residue 94, that is [91-96], was not observed in any of the analyses. (It is possible that an SRM approach may identify the relevant peptide more reproducibly; however, the Orbitrap instrument used in this work is not suited to such an approach). One of the analyses following extraction with ammonium bicarbonate resulted in the identification of the missed-cleavage tryptic peptide [81-96] (m/zmeas 1791.9931, m/zcalc 1791.9851, Δ 4.9 ppm) from FABP1. The CID mass spectrum obtained from the [M+3H]3+ peptide ions is shown in Figure 2a. The fragmentation sequence coverage for the peptide is poor with no cleavage around the potential substitution site. Clearly, as the variant is the result of a single amino acid substitution and the sequences of FAPB1 and its variant are virtually identical, with the exception of peptide [81-96], the peptides assigned to FABP1 in the database search could also have originated from the variant. The human SwissProt database against which the database was searched was supplemented with the FABP1 variant (hereafter referred to as FABP1TA). The peptide [90-96] from FABP1TA was not identified in any of the analyses; however, the missed-cleavage peptide [81-96] was identified in one of the ammonium bicarbonate extraction replicates (m/zmeas 1761.9831, m/zcalc 1761.9745, Δ 4.5 ppm). The CID spectrum is shown in Figure 2b. Again, fragmentation sequence coverage is poor and no fragmentation was observed adjacent to the site of substitution. It should be noted that top-down analysis of this tissue sample (see below) revealed the presence of the variant.Figure 2

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