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Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards.

Kramer G, Woolerton Y, van Straalen JP, Vissers JP, Dekker N, Langridge JI, Beynon RJ, Speijer D, Sturk A, Aerts JM - PLoS ONE (2015)

Bottom Line: Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research.Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL-40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347).This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands.

ABSTRACT

Background: Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research.

Results: Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL-40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347). Quantitative results were obtained by label-free mass spectrometry using a single internal standard to estimate protein concentrations. This approach resulted in quantitative results for 59 proteins (cut off ≥11 samples quantified) of which 41 proteins were quantified in all 31 samples and 23 of these with an inter-assay variability of ≤ 20%. Results for 7 apolipoproteins were compared with those obtained using isotope-labelled standards, while 12 proteins were compared to routine immunoassays. Comparison of quantitative data obtained by LCMSE and immunoassays showed good to excellent correlations in relative protein abundance (r = 0.72-0.96) and comparable median concentrations for 8 out of 12 proteins tested. Plasma concentrations of 56 proteins determined by LCMSE were of similar accuracy as those reported by targeted studies and 7 apolipoproteins quantified by isotope-labelled standards, when compared to reference concentrations from literature.

Conclusions: This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins.

No MeSH data available.


Quantitation of plasma protein concentrations by HI3, QconCAT and immunoassay.(a) Intra-assay variation of apolipoproteins by HI3 and QconCAT on a single day of measurements. (b) Protein concentration quantified by HI3 peptide quantitation (squares) or QconCAT (circles) and their reference ranges (grey boxes) in 5 pooled plasma samples. (c) Median HI3 peptide quantitation (squares) in 31 samples apart from fibrinogen (n = 30) and complement C4 (n = 29), error bars indicate the minimal and maximum value measured in the plasma samples, while grey boxes indicate ranges quantified by immunoassays in the same samples.
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pone.0140097.g002: Quantitation of plasma protein concentrations by HI3, QconCAT and immunoassay.(a) Intra-assay variation of apolipoproteins by HI3 and QconCAT on a single day of measurements. (b) Protein concentration quantified by HI3 peptide quantitation (squares) or QconCAT (circles) and their reference ranges (grey boxes) in 5 pooled plasma samples. (c) Median HI3 peptide quantitation (squares) in 31 samples apart from fibrinogen (n = 30) and complement C4 (n = 29), error bars indicate the minimal and maximum value measured in the plasma samples, while grey boxes indicate ranges quantified by immunoassays in the same samples.

Mentions: Next we compared concentrations quantified by HI3 peptide quantitation and stable isotope-labelled standards. For this we used an artificially constructed QconCAT protein expressed in E. coli to introduce stable isotope-labelled lysine and arginine residues. This QconCAT [43], is a concatamer of two proteotypic peptides per protein for 11 apolipoproteins. It is expressed and purified by Ni-column chromatography and was quantified by BCA-assay to ascertain the protein concentration (see materials and methods). The QconCAT was spiked into 5 pooled plasma samples within the linear response range (see S4 Fig). Following tryptic digestion and LCMSE analysis, extracted ion intensities of endogenous (apolipoprotein-derived) and isotope-labelled peptides (QconCAT-derived) we could ascertain plasma protein concentrations for seven apolipoproteins (Fig 2, materials and methods). The Intra-assay variability (IAV) of quantities obtained for apolipoproteins using a QconCAT internal standard are generally lower than those obtained by HI3 quantitation (Fig 2a). Overall the apolipoprotein concentrations quantified by QconCAT are two-fold (median: 1.9, range: 1.3–3.2 fold, see S12 Table) higher than those quantified by HI3 quantitation. As digestion and measuring conditions were identical for these samples (QconCAT and HI3 quantitation was done within the same pooled plasma samples), a likely reason for this offset comes from a difference in the actual amount in fmol added of one or both standards and the value(s) used for calculation of the concentrations. As mentioned in the materials and methods, the concentration of the QconCAT was estimated by BCA-assay, while MASSPREP protein digestion standard amounts of ENO1 and ADH1 are given by the manufacturer. Another possible explanation, assuming spiking in of both standards was accurate, would be a slower release of QconCAT heavy labelled peptides compared to endogenous light peptides from the apolipoproteins which would result in a relative overestimation of concentrations. However, overall QconCATs seem to be subjected to fast and complete digestion [36]. Notwithstanding the differences in concentrations quantified, QconCAT quantitation of Apo A-IV and C-II fall within their reference ranges, whereas Apo A-II, B-100, C-III and E are less than a factor of 2 out of their reference ranges. Only the concentration of ApoA1 is more than a factor 2 out of the reference range. With HI3 quantitation, Apo A-II, Apo E are less than a factor of 2 outside their reference range, while Apo C-II is more than a factor two out of range. Overall, HI3 quantitation seems to be as close to reference ranges in plasma as the QconCAT internal standard for the apolipoproteins detected (Fig 2b).


Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards.

Kramer G, Woolerton Y, van Straalen JP, Vissers JP, Dekker N, Langridge JI, Beynon RJ, Speijer D, Sturk A, Aerts JM - PLoS ONE (2015)

Quantitation of plasma protein concentrations by HI3, QconCAT and immunoassay.(a) Intra-assay variation of apolipoproteins by HI3 and QconCAT on a single day of measurements. (b) Protein concentration quantified by HI3 peptide quantitation (squares) or QconCAT (circles) and their reference ranges (grey boxes) in 5 pooled plasma samples. (c) Median HI3 peptide quantitation (squares) in 31 samples apart from fibrinogen (n = 30) and complement C4 (n = 29), error bars indicate the minimal and maximum value measured in the plasma samples, while grey boxes indicate ranges quantified by immunoassays in the same samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608811&req=5

pone.0140097.g002: Quantitation of plasma protein concentrations by HI3, QconCAT and immunoassay.(a) Intra-assay variation of apolipoproteins by HI3 and QconCAT on a single day of measurements. (b) Protein concentration quantified by HI3 peptide quantitation (squares) or QconCAT (circles) and their reference ranges (grey boxes) in 5 pooled plasma samples. (c) Median HI3 peptide quantitation (squares) in 31 samples apart from fibrinogen (n = 30) and complement C4 (n = 29), error bars indicate the minimal and maximum value measured in the plasma samples, while grey boxes indicate ranges quantified by immunoassays in the same samples.
Mentions: Next we compared concentrations quantified by HI3 peptide quantitation and stable isotope-labelled standards. For this we used an artificially constructed QconCAT protein expressed in E. coli to introduce stable isotope-labelled lysine and arginine residues. This QconCAT [43], is a concatamer of two proteotypic peptides per protein for 11 apolipoproteins. It is expressed and purified by Ni-column chromatography and was quantified by BCA-assay to ascertain the protein concentration (see materials and methods). The QconCAT was spiked into 5 pooled plasma samples within the linear response range (see S4 Fig). Following tryptic digestion and LCMSE analysis, extracted ion intensities of endogenous (apolipoprotein-derived) and isotope-labelled peptides (QconCAT-derived) we could ascertain plasma protein concentrations for seven apolipoproteins (Fig 2, materials and methods). The Intra-assay variability (IAV) of quantities obtained for apolipoproteins using a QconCAT internal standard are generally lower than those obtained by HI3 quantitation (Fig 2a). Overall the apolipoprotein concentrations quantified by QconCAT are two-fold (median: 1.9, range: 1.3–3.2 fold, see S12 Table) higher than those quantified by HI3 quantitation. As digestion and measuring conditions were identical for these samples (QconCAT and HI3 quantitation was done within the same pooled plasma samples), a likely reason for this offset comes from a difference in the actual amount in fmol added of one or both standards and the value(s) used for calculation of the concentrations. As mentioned in the materials and methods, the concentration of the QconCAT was estimated by BCA-assay, while MASSPREP protein digestion standard amounts of ENO1 and ADH1 are given by the manufacturer. Another possible explanation, assuming spiking in of both standards was accurate, would be a slower release of QconCAT heavy labelled peptides compared to endogenous light peptides from the apolipoproteins which would result in a relative overestimation of concentrations. However, overall QconCATs seem to be subjected to fast and complete digestion [36]. Notwithstanding the differences in concentrations quantified, QconCAT quantitation of Apo A-IV and C-II fall within their reference ranges, whereas Apo A-II, B-100, C-III and E are less than a factor of 2 out of their reference ranges. Only the concentration of ApoA1 is more than a factor 2 out of the reference range. With HI3 quantitation, Apo A-II, Apo E are less than a factor of 2 outside their reference range, while Apo C-II is more than a factor two out of range. Overall, HI3 quantitation seems to be as close to reference ranges in plasma as the QconCAT internal standard for the apolipoproteins detected (Fig 2b).

Bottom Line: Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research.Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL-40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347).This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands.

ABSTRACT

Background: Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research.

Results: Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL-40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347). Quantitative results were obtained by label-free mass spectrometry using a single internal standard to estimate protein concentrations. This approach resulted in quantitative results for 59 proteins (cut off ≥11 samples quantified) of which 41 proteins were quantified in all 31 samples and 23 of these with an inter-assay variability of ≤ 20%. Results for 7 apolipoproteins were compared with those obtained using isotope-labelled standards, while 12 proteins were compared to routine immunoassays. Comparison of quantitative data obtained by LCMSE and immunoassays showed good to excellent correlations in relative protein abundance (r = 0.72-0.96) and comparable median concentrations for 8 out of 12 proteins tested. Plasma concentrations of 56 proteins determined by LCMSE were of similar accuracy as those reported by targeted studies and 7 apolipoproteins quantified by isotope-labelled standards, when compared to reference concentrations from literature.

Conclusions: This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins.

No MeSH data available.