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Plasma level of C-terminal peptides and polymeric AAT in pediatric samples. EDTA-plasma was analyzed by LC-MS/MS for AAT peptide analysis or by Western Blot for polymeric AAT. (A) The three peptides C36, C37, and C42 could be consistently found at micromolar concentrations in all 20 samples of pediatric Pi*ZZ carrier. Peptide level below the methods lower quantification limit (LLOQ = 0.01 µM for C37 or 0.025 µM for C36 and C42) were imputed by dividing LLOQ by the square root of 2 ( n = 4 for C37). (B) Representation of the concentration (mg/dL) of AAT polymers among the 20 pediatric AATD patients. Boxplots represent the median and interquartile range with whiskers indicating the lowest and highest values. The median concentration (in µM for AAT peptide and mg/dL for AAT polymers) is indicated above each individual analyte. (C) Plasma samples were separated on native 7.5% <t>polyacrylamide</t> gels, blotted onto PVDF membranes and polymeric AAT was visualized using the LG96 monoclonal mouse antibody. Z-AAT purified from human plasma (1.5 µg/mL) was loaded onto each gel as an internal standard for semiquantitative densitometric analysis. Representative Western Blot images are shown. AAT, alpha-1 antitrypsin.
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Plasma level of C-terminal peptides and polymeric AAT in pediatric samples. EDTA-plasma was analyzed by LC-MS/MS for AAT peptide analysis or by Western Blot for polymeric AAT. (A) The three peptides C36, C37, and C42 could be consistently found at micromolar concentrations in all 20 samples of pediatric Pi*ZZ carrier. Peptide level below the methods lower quantification limit (LLOQ = 0.01 µM for C37 or 0.025 µM for C36 and C42) were imputed by dividing LLOQ by the square root of 2 ( n = 4 for C37). (B) Representation of the concentration (mg/dL) of AAT polymers among the 20 pediatric AATD patients. Boxplots represent the median and interquartile range with whiskers indicating the lowest and highest values. The median concentration (in µM for AAT peptide and mg/dL for AAT polymers) is indicated above each individual analyte. (C) Plasma samples were separated on native 7.5% <t>polyacrylamide</t> gels, blotted onto PVDF membranes and polymeric AAT was visualized using the LG96 monoclonal mouse antibody. Z-AAT purified from human plasma (1.5 µg/mL) was loaded onto each gel as an internal standard for semiquantitative densitometric analysis. Representative Western Blot images are shown. AAT, alpha-1 antitrypsin.
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Plasma level of C-terminal peptides and polymeric AAT in pediatric samples. EDTA-plasma was analyzed by LC-MS/MS for AAT peptide analysis or by Western Blot for polymeric AAT. (A) The three peptides C36, C37, and C42 could be consistently found at micromolar concentrations in all 20 samples of pediatric Pi*ZZ carrier. Peptide level below the methods lower quantification limit (LLOQ = 0.01 µM for C37 or 0.025 µM for C36 and C42) were imputed by dividing LLOQ by the square root of 2 ( n = 4 for C37). (B) Representation of the concentration (mg/dL) of AAT polymers among the 20 pediatric AATD patients. Boxplots represent the median and interquartile range with whiskers indicating the lowest and highest values. The median concentration (in µM for AAT peptide and mg/dL for AAT polymers) is indicated above each individual analyte. (C) Plasma samples were separated on native 7.5% <t>polyacrylamide</t> gels, blotted onto PVDF membranes and polymeric AAT was visualized using the LG96 monoclonal mouse antibody. Z-AAT purified from human plasma (1.5 µg/mL) was loaded onto each gel as an internal standard for semiquantitative densitometric analysis. Representative Western Blot images are shown. AAT, alpha-1 antitrypsin.
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Plasma level of C-terminal peptides and polymeric AAT in pediatric samples. EDTA-plasma was analyzed by LC-MS/MS for AAT peptide analysis or by Western Blot for polymeric AAT. (A) The three peptides C36, C37, and C42 could be consistently found at micromolar concentrations in all 20 samples of pediatric Pi*ZZ carrier. Peptide level below the methods lower quantification limit (LLOQ = 0.01 µM for C37 or 0.025 µM for C36 and C42) were imputed by dividing LLOQ by the square root of 2 ( n = 4 for C37). (B) Representation of the concentration (mg/dL) of AAT polymers among the 20 pediatric AATD patients. Boxplots represent the median and interquartile range with whiskers indicating the lowest and highest values. The median concentration (in µM for AAT peptide and mg/dL for AAT polymers) is indicated above each individual analyte. (C) Plasma samples were separated on native 7.5% <t>polyacrylamide</t> gels, blotted onto PVDF membranes and polymeric AAT was visualized using the LG96 monoclonal mouse antibody. Z-AAT purified from human plasma (1.5 µg/mL) was loaded onto each gel as an internal standard for semiquantitative densitometric analysis. Representative Western Blot images are shown. AAT, alpha-1 antitrypsin.
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Plasma level of C-terminal peptides and polymeric AAT in pediatric samples. EDTA-plasma was analyzed by LC-MS/MS for AAT peptide analysis or by Western Blot for polymeric AAT. (A) The three peptides C36, C37, and C42 could be consistently found at micromolar concentrations in all 20 samples of pediatric Pi*ZZ carrier. Peptide level below the methods lower quantification limit (LLOQ = 0.01 µM for C37 or 0.025 µM for C36 and C42) were imputed by dividing LLOQ by the square root of 2 ( n = 4 for C37). (B) Representation of the concentration (mg/dL) of AAT polymers among the 20 pediatric AATD patients. Boxplots represent the median and interquartile range with whiskers indicating the lowest and highest values. The median concentration (in µM for AAT peptide and mg/dL for AAT polymers) is indicated above each individual analyte. (C) Plasma samples were separated on native 7.5% <t>polyacrylamide</t> gels, blotted onto PVDF membranes and polymeric AAT was visualized using the LG96 monoclonal mouse antibody. Z-AAT purified from human plasma (1.5 µg/mL) was loaded onto each gel as an internal standard for semiquantitative densitometric analysis. Representative Western Blot images are shown. AAT, alpha-1 antitrypsin.
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bis tris acrylamide gel  (Thermo Fisher)
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Plasma level of C-terminal peptides and polymeric AAT in pediatric samples. EDTA-plasma was analyzed by LC-MS/MS for AAT peptide analysis or by Western Blot for polymeric AAT. (A) The three peptides C36, C37, and C42 could be consistently found at micromolar concentrations in all 20 samples of pediatric Pi*ZZ carrier. Peptide level below the methods lower quantification limit (LLOQ = 0.01 µM for C37 or 0.025 µM for C36 and C42) were imputed by dividing LLOQ by the square root of 2 ( n = 4 for C37). (B) Representation of the concentration (mg/dL) of AAT polymers among the 20 pediatric AATD patients. Boxplots represent the median and interquartile range with whiskers indicating the lowest and highest values. The median concentration (in µM for AAT peptide and mg/dL for AAT polymers) is indicated above each individual analyte. (C) Plasma samples were separated on native 7.5% <t>polyacrylamide</t> gels, blotted onto PVDF membranes and polymeric AAT was visualized using the LG96 monoclonal mouse antibody. Z-AAT purified from human plasma (1.5 µg/mL) was loaded onto each gel as an internal standard for semiquantitative densitometric analysis. Representative Western Blot images are shown. AAT, alpha-1 antitrypsin.
Bis Tris Acrylamide Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Plasma level of C-terminal peptides and polymeric AAT in pediatric samples. EDTA-plasma was analyzed by LC-MS/MS for AAT peptide analysis or by Western Blot for polymeric AAT. (A) The three peptides C36, C37, and C42 could be consistently found at micromolar concentrations in all 20 samples of pediatric Pi*ZZ carrier. Peptide level below the methods lower quantification limit (LLOQ = 0.01 µM for C37 or 0.025 µM for C36 and C42) were imputed by dividing LLOQ by the square root of 2 ( n = 4 for C37). (B) Representation of the concentration (mg/dL) of AAT polymers among the 20 pediatric AATD patients. Boxplots represent the median and interquartile range with whiskers indicating the lowest and highest values. The median concentration (in µM for AAT peptide and mg/dL for AAT polymers) is indicated above each individual analyte. (C) Plasma samples were separated on native 7.5% polyacrylamide gels, blotted onto PVDF membranes and polymeric AAT was visualized using the LG96 monoclonal mouse antibody. Z-AAT purified from human plasma (1.5 µg/mL) was loaded onto each gel as an internal standard for semiquantitative densitometric analysis. Representative Western Blot images are shown. AAT, alpha-1 antitrypsin.

Journal: Frontiers in Pediatrics

Article Title: Circulating C-terminal peptides and polymers of alpha-1 antitrypsin as putative markers of pediatric Pi*ZZ liver disease

doi: 10.3389/fped.2025.1717317

Figure Lengend Snippet: Plasma level of C-terminal peptides and polymeric AAT in pediatric samples. EDTA-plasma was analyzed by LC-MS/MS for AAT peptide analysis or by Western Blot for polymeric AAT. (A) The three peptides C36, C37, and C42 could be consistently found at micromolar concentrations in all 20 samples of pediatric Pi*ZZ carrier. Peptide level below the methods lower quantification limit (LLOQ = 0.01 µM for C37 or 0.025 µM for C36 and C42) were imputed by dividing LLOQ by the square root of 2 ( n = 4 for C37). (B) Representation of the concentration (mg/dL) of AAT polymers among the 20 pediatric AATD patients. Boxplots represent the median and interquartile range with whiskers indicating the lowest and highest values. The median concentration (in µM for AAT peptide and mg/dL for AAT polymers) is indicated above each individual analyte. (C) Plasma samples were separated on native 7.5% polyacrylamide gels, blotted onto PVDF membranes and polymeric AAT was visualized using the LG96 monoclonal mouse antibody. Z-AAT purified from human plasma (1.5 µg/mL) was loaded onto each gel as an internal standard for semiquantitative densitometric analysis. Representative Western Blot images are shown. AAT, alpha-1 antitrypsin.

Article Snippet: Samples were separated on 7.5% native polyacrylamide gels (40% Acrylamide/Bis Solution 29:1, Bio-Rad Laboratories Inc., Hercules, CA, USA) and subsequently transferred onto polyvinylidene fluoride (PVDF, Merck Millipore, Merck KGaA, Darmstadt) membranes.

Techniques: Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Western Blot, Concentration Assay, Purification