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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.


Related in: MedlinePlus

Correlation of concentrations obtained by either label free mass spectrometry or clinical immunoassay of 12 abundant plasma proteins.Each dot represents the correlation between the protein concentration (g/L) of a single sample determined by clinical immunoassays (x-axis) and label free mass spectrometry (y-axis). Regression lines (solid black) obtained by ordinary least squares linear regression and the formulas describing them are shown. The dashed line, x = y, represents perfect correlation for comparison.
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pone.0140097.g003: Correlation of concentrations obtained by either label free mass spectrometry or clinical immunoassay of 12 abundant plasma proteins.Each dot represents the correlation between the protein concentration (g/L) of a single sample determined by clinical immunoassays (x-axis) and label free mass spectrometry (y-axis). Regression lines (solid black) obtained by ordinary least squares linear regression and the formulas describing them are shown. The dashed line, x = y, represents perfect correlation for comparison.

Mentions: We also compared the concentrations determined by HI3 peptide quantitation with those of routine clinical immunoassays in the same sample set (31 samples), which are currently the standard for plasma protein determination in clinical practice. For the 12 proteins tested, eight concentrations determined by HI3 peptide quantitation fall within the range determined by immunoassay in the same set, whereas three proteins are not more than a factor of two outside of it (Fig 2c). Only in the case of complement C4 the concentration determined by LCMSE was markedly outside the immunoassay range (see Table 4 and Fig 2c). Protein concentration data obtained by LCMSE were also compared to those obtained by immunoassay in individual samples (Fig 3). Ordinary least squares linear regression reveals linear relationships. Spearman correlation coefficients, allowing the detection of covariation in the assays, are given in Table 4. The majority of protein concentrations determined by LCMSE showed good (r = 0.8–0.9) or very good correlation (r >0.9) with immunoassays. In the case of three proteins correlation is fair (>0.7). Reproducibility of immunoassays was found to be better for each of the 12 proteins examined. The median inter assay variance for HI3 peptide quantitation was 19% (12–30%) compared to 3% (1–5%) for immunoassays (Table 4).


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)

Correlation of concentrations obtained by either label free mass spectrometry or clinical immunoassay of 12 abundant plasma proteins.Each dot represents the correlation between the protein concentration (g/L) of a single sample determined by clinical immunoassays (x-axis) and label free mass spectrometry (y-axis). Regression lines (solid black) obtained by ordinary least squares linear regression and the formulas describing them are shown. The dashed line, x = y, represents perfect correlation for comparison.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140097.g003: Correlation of concentrations obtained by either label free mass spectrometry or clinical immunoassay of 12 abundant plasma proteins.Each dot represents the correlation between the protein concentration (g/L) of a single sample determined by clinical immunoassays (x-axis) and label free mass spectrometry (y-axis). Regression lines (solid black) obtained by ordinary least squares linear regression and the formulas describing them are shown. The dashed line, x = y, represents perfect correlation for comparison.
Mentions: We also compared the concentrations determined by HI3 peptide quantitation with those of routine clinical immunoassays in the same sample set (31 samples), which are currently the standard for plasma protein determination in clinical practice. For the 12 proteins tested, eight concentrations determined by HI3 peptide quantitation fall within the range determined by immunoassay in the same set, whereas three proteins are not more than a factor of two outside of it (Fig 2c). Only in the case of complement C4 the concentration determined by LCMSE was markedly outside the immunoassay range (see Table 4 and Fig 2c). Protein concentration data obtained by LCMSE were also compared to those obtained by immunoassay in individual samples (Fig 3). Ordinary least squares linear regression reveals linear relationships. Spearman correlation coefficients, allowing the detection of covariation in the assays, are given in Table 4. The majority of protein concentrations determined by LCMSE showed good (r = 0.8–0.9) or very good correlation (r >0.9) with immunoassays. In the case of three proteins correlation is fair (>0.7). Reproducibility of immunoassays was found to be better for each of the 12 proteins examined. The median inter assay variance for HI3 peptide quantitation was 19% (12–30%) compared to 3% (1–5%) for immunoassays (Table 4).

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.


Related in: MedlinePlus