Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Nov;20(11):2006-12.
doi: 10.1016/j.jasms.2009.07.019. Epub 2009 Aug 7.

Evaluation of the ALiPHAT method for PC-IDMS and correlation of limits-of-detection with nonpolar surface area

D Keith Williams Jr  1 , Daniel L Comins, Jerry L Whitten, David C Muddiman
Affiliations

Evaluation of the ALiPHAT method for PC-IDMS and correlation of limits-of-detection with nonpolar surface area

D Keith Williams Jr et al. J Am Soc Mass Spectrom. 2009 Nov.

Abstract

PC-IDMS experiments for two peptides, laminin nonapeptide and the N-terminal tryptic peptide of prostate specific antigen, were performed utilizing a variety of alkylating reagents. These experiments were conducted to investigate how hydrophobicity influences the limits-of-detection (LOD) by altering their electrospray ionization response. Nonpolar surface areas were calculated for both peptides and all alkylating reagents to provide an estimate of the hydrophobicity of the differently alkylated peptides. Decreases in LOD by 2-fold were observed for both peptides between the best and worst performing combination of alkylating reagent. However, while an increase in hydrophobicity was found to aid in decreasing LOD to an extent, beyond a certain hydrophobicity, we observed a decrease.

PubMed Disclaimer

Figures

Figure 1
Figure 1
A) Illustrates the amino acid sequences of the two peptides used in this study, the transition used by the mass spectrometer, and the nonpolar surface area for the given peptide. The underlined amino acid shows the stable isotope labeled form for in the internal standard. B) The alkylating reagents utilized in this study are shown along with their abbreviation used in this manuscript and their nonpolar surface area.
Figure 2
Figure 2
Calibration curves for laminin and tpPSA1–9 for both gradient elution and isocratic elution experiments are shown. The overlap of the peptides with different alkylating reagents demonstrated that all combination could be used to perform absolute quantification experiments with a PC-IDMS methodology.
Figure 3
Figure 3
Sensitivity is shown to differ for both peptides depending on which alkylating reagent is utilized. Abbreviations denote which alkylating reagent was used. Inset is the extracted ion chromatogram for the best and worst performing peptide and alkylating reagents for the denoted experimental set.
Figure 4
Figure 4
The relationship between normalized nonpolar surface area and ESI response is shown for A) Gradient elution and B) Isocratic elution of 5 fmole on column. Squares represent laminin, while triangles represent tpPSA1–9.
See this image and copyright information in PMC

References

    1. Barr JR, Maggio VL, Patterson DG, et al. Isotope dilution mass spectrometric quantification of specific proteins: Model application with apolipoprotein A-I. Clin. Chem. 1996;42(10):1676–1682. - PubMed
    1. de Leenheer AP, Thienpont LM. Applications of Isotope-Dilution Mass-Spectrometry in Clinical-Chemistry, Pharmacokinetics, and Toxicology. Mass Spectrom. Rev. 1992;11(4):249–307.
    1. Gygi SP, Rist B, Gerber SA. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 1999;17(10):994–999. - PubMed
    1. Zhou HL, Ranish JA, Watts JD, et al. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nature Biotechnology. 2002;20(5):512–515. - PubMed
    1. Li JX, Steen H, Gygi SP. Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents - The yeast salinity stress response. Mol. Cell. Proteomics. 2003;2(11):1198–1204. - PubMed

Publication types