Abstract
Quantitation of low-abundance protein modifications involves significant analytical challenges, especially in biologically important applications, such as studying the role of post-translational modifications in biology and measurement of the effects of reactive drug metabolites. 14C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time 14C quantitation of high-performance liquid chromatography (HPLC) separations by liquid sample accelerator mass spectrometry (LS-AMS). By enabling direct HPLC-AMS coupling, LS-AMS overcomes several major limitations of conventional HPLC-AMS, where individual HPLC fractions must be collected and converted to graphite before measurement. To demonstrate LS-AMS and compare the new technology to traditional solid sample AMS (SS-AMS), reduced and native bovine serum albumin (BSA) was modified by 14C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 106 to 108 proteins. 14C incorporated into modified BSA was measured by solid carbon AMS and LS-AMS. BSA peptides were generated by tryptic digestion. Analysis of HPLC-separated peptides was performed in parallel by LS-AMS, fraction collection combined with SS-AMS, and (for peptide identification) electrospray ionization and tandem mass spectrometry (ESI-MS/MS). LS-AMS enabled 14C quantitation from ng sample sizes and was 100 times more sensitive to 14C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol 14C/peak. Additionally, LS-AMS turnaround times were minutes instead of days, and HPLC trace analyses required 1/6th the AMS instrument time required for analysis of graphite fractions by SS-AMS.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3644-3650 |
| Number of pages | 7 |
| Journal | Analytical Chemistry |
| Volume | 85 |
| Issue number | 7 |
| DOIs | |
| State | Published - Apr 2 2013 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Analytical Chemistry
Cite this
Directly coupled high-performance liquid chromatography-accelerator mass spectrometry measurement of chemically modified protein and peptides. / Thomas, Avi T.; Stewart, Benjamin J.; Ognibene, Ted J.; Turteltaub, Ken W; Bench, Graham.
In: Analytical Chemistry, Vol. 85, No. 7, 02.04.2013, p. 3644-3650.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Directly coupled high-performance liquid chromatography-accelerator mass spectrometry measurement of chemically modified protein and peptides
AU - Thomas, Avi T.
AU - Stewart, Benjamin J.
AU - Ognibene, Ted J.
AU - Turteltaub, Ken W
AU - Bench, Graham
PY - 2013/4/2
Y1 - 2013/4/2
N2 - Quantitation of low-abundance protein modifications involves significant analytical challenges, especially in biologically important applications, such as studying the role of post-translational modifications in biology and measurement of the effects of reactive drug metabolites. 14C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time 14C quantitation of high-performance liquid chromatography (HPLC) separations by liquid sample accelerator mass spectrometry (LS-AMS). By enabling direct HPLC-AMS coupling, LS-AMS overcomes several major limitations of conventional HPLC-AMS, where individual HPLC fractions must be collected and converted to graphite before measurement. To demonstrate LS-AMS and compare the new technology to traditional solid sample AMS (SS-AMS), reduced and native bovine serum albumin (BSA) was modified by 14C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 106 to 108 proteins. 14C incorporated into modified BSA was measured by solid carbon AMS and LS-AMS. BSA peptides were generated by tryptic digestion. Analysis of HPLC-separated peptides was performed in parallel by LS-AMS, fraction collection combined with SS-AMS, and (for peptide identification) electrospray ionization and tandem mass spectrometry (ESI-MS/MS). LS-AMS enabled 14C quantitation from ng sample sizes and was 100 times more sensitive to 14C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol 14C/peak. Additionally, LS-AMS turnaround times were minutes instead of days, and HPLC trace analyses required 1/6th the AMS instrument time required for analysis of graphite fractions by SS-AMS.
AB - Quantitation of low-abundance protein modifications involves significant analytical challenges, especially in biologically important applications, such as studying the role of post-translational modifications in biology and measurement of the effects of reactive drug metabolites. 14C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time 14C quantitation of high-performance liquid chromatography (HPLC) separations by liquid sample accelerator mass spectrometry (LS-AMS). By enabling direct HPLC-AMS coupling, LS-AMS overcomes several major limitations of conventional HPLC-AMS, where individual HPLC fractions must be collected and converted to graphite before measurement. To demonstrate LS-AMS and compare the new technology to traditional solid sample AMS (SS-AMS), reduced and native bovine serum albumin (BSA) was modified by 14C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 106 to 108 proteins. 14C incorporated into modified BSA was measured by solid carbon AMS and LS-AMS. BSA peptides were generated by tryptic digestion. Analysis of HPLC-separated peptides was performed in parallel by LS-AMS, fraction collection combined with SS-AMS, and (for peptide identification) electrospray ionization and tandem mass spectrometry (ESI-MS/MS). LS-AMS enabled 14C quantitation from ng sample sizes and was 100 times more sensitive to 14C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol 14C/peak. Additionally, LS-AMS turnaround times were minutes instead of days, and HPLC trace analyses required 1/6th the AMS instrument time required for analysis of graphite fractions by SS-AMS.
UR - http://www.scopus.com/inward/record.url?scp=84875795023&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84875795023&partnerID=8YFLogxK
U2 - 10.1021/ac303609n
DO - 10.1021/ac303609n
M3 - Article
C2 - 23413773
AN - SCOPUS:84875795023
VL - 85
SP - 3644
EP - 3650
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 7
ER -