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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

HI3 peptide quantitation with a single protein digest standard and digest standard comparison.(a) Summed signal intensity of the protein digest standard ENO1 (grey square) and ADH1 (dark grey circle) added at increasing concentrations to a plasma digest. (b) Quantitation of albumin using either ENO1 or ADH1 as the internal standard in 17 indivual samples. The regression line (solid black) and its formula, obtained by ordinary least squares linear regression, is depicted, with the dashed line representing perfect correlation. (c) 57 proteins from Table 2 for which reference ranges from literature were available, are ordered according to their median concentration determined by HI3 peptide quantitation (dark grey squares, quantified in ≥ 11 out of 31 samples). Error bars indicate the minimal and maximum value measured in the plasma samples. The reference ranges (grey boxes) are taken from Hortin et al. [42]. Protein no. correspond to the numbers given in Table 2.
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pone.0140097.g001: HI3 peptide quantitation with a single protein digest standard and digest standard comparison.(a) Summed signal intensity of the protein digest standard ENO1 (grey square) and ADH1 (dark grey circle) added at increasing concentrations to a plasma digest. (b) Quantitation of albumin using either ENO1 or ADH1 as the internal standard in 17 indivual samples. The regression line (solid black) and its formula, obtained by ordinary least squares linear regression, is depicted, with the dashed line representing perfect correlation. (c) 57 proteins from Table 2 for which reference ranges from literature were available, are ordered according to their median concentration determined by HI3 peptide quantitation (dark grey squares, quantified in ≥ 11 out of 31 samples). Error bars indicate the minimal and maximum value measured in the plasma samples. The reference ranges (grey boxes) are taken from Hortin et al. [42]. Protein no. correspond to the numbers given in Table 2.

Mentions: Median (ng/mL): median of protein concentrations determined from number of samples shown in column n. IRV: inter-assay variation, the coefficients of variation obtained from 7 aliquots of a pooled sample separately digested and measured over the course of 3 months of normal operation of the instrument. IAV: intra-assay variation, coefficient of variation obtained from 6 aliquots of a pooled sample separately digested and subsequently measured during 1 day. AV: analytical variability, determined from 10 replicate injections of a single digested sample throughout 9 days of measurements. LIN: linearity of measurements, the Pearson’s correlation coefficient shows linearity between total protein load on the analytical column and nanogram protein quantified by HI3 peptide quantitation using the ADH1 digest standard; only determined when at least 4 points were available for a protein. See also S1 Fig. ST: Pearson’s correlation of protein quantitation using two different digest standards (ADH1 and ENO1 from yeast). LOQ: limit of quantitation (ng/mL) estimated by dilution of a plasma sample in a constant background of a digest standard. n: number of samples (out of 31) in which the protein was quantified, FN: protein number on the x-axis of Fig 1c. hc: heavy chain.


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)

HI3 peptide quantitation with a single protein digest standard and digest standard comparison.(a) Summed signal intensity of the protein digest standard ENO1 (grey square) and ADH1 (dark grey circle) added at increasing concentrations to a plasma digest. (b) Quantitation of albumin using either ENO1 or ADH1 as the internal standard in 17 indivual samples. The regression line (solid black) and its formula, obtained by ordinary least squares linear regression, is depicted, with the dashed line representing perfect correlation. (c) 57 proteins from Table 2 for which reference ranges from literature were available, are ordered according to their median concentration determined by HI3 peptide quantitation (dark grey squares, quantified in ≥ 11 out of 31 samples). Error bars indicate the minimal and maximum value measured in the plasma samples. The reference ranges (grey boxes) are taken from Hortin et al. [42]. Protein no. correspond to the numbers given in Table 2.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140097.g001: HI3 peptide quantitation with a single protein digest standard and digest standard comparison.(a) Summed signal intensity of the protein digest standard ENO1 (grey square) and ADH1 (dark grey circle) added at increasing concentrations to a plasma digest. (b) Quantitation of albumin using either ENO1 or ADH1 as the internal standard in 17 indivual samples. The regression line (solid black) and its formula, obtained by ordinary least squares linear regression, is depicted, with the dashed line representing perfect correlation. (c) 57 proteins from Table 2 for which reference ranges from literature were available, are ordered according to their median concentration determined by HI3 peptide quantitation (dark grey squares, quantified in ≥ 11 out of 31 samples). Error bars indicate the minimal and maximum value measured in the plasma samples. The reference ranges (grey boxes) are taken from Hortin et al. [42]. Protein no. correspond to the numbers given in Table 2.
Mentions: Median (ng/mL): median of protein concentrations determined from number of samples shown in column n. IRV: inter-assay variation, the coefficients of variation obtained from 7 aliquots of a pooled sample separately digested and measured over the course of 3 months of normal operation of the instrument. IAV: intra-assay variation, coefficient of variation obtained from 6 aliquots of a pooled sample separately digested and subsequently measured during 1 day. AV: analytical variability, determined from 10 replicate injections of a single digested sample throughout 9 days of measurements. LIN: linearity of measurements, the Pearson’s correlation coefficient shows linearity between total protein load on the analytical column and nanogram protein quantified by HI3 peptide quantitation using the ADH1 digest standard; only determined when at least 4 points were available for a protein. See also S1 Fig. ST: Pearson’s correlation of protein quantitation using two different digest standards (ADH1 and ENO1 from yeast). LOQ: limit of quantitation (ng/mL) estimated by dilution of a plasma sample in a constant background of a digest standard. n: number of samples (out of 31) in which the protein was quantified, FN: protein number on the x-axis of Fig 1c. hc: heavy chain.

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