Feasibility Assessment of a Bioanalytical Method for Quantification of a 14.3 kDa Protein in Human Plasma using Tryptic Digestion LC-MS/MS without a Requirement for Antibodies

Mar 09 2010

Author: Matthew F Ewles, Mohammed Abrar, Lee Goodwin* and David Bakes - Covance Laboratories Ltd. on behalf of Unassigned Independent Article

Free to read

This article has been unlocked and is ready to read.


A bioanalytical method for the quantification of a 14.3 kDa protein in human plasma has been developed using proteolytic digestion and LC-MS/MS quantification of a surrogate peptide. The technique provides an approach for the quantification of small protein therapeutics in biological fluids without the need for antibodies. Conventional LC-MS/MS sample handling or analysis procedures are not suitable for molecules of this size, and immunoassay methods may not yield adequate selectivity or sensitivity. Data is presented that demonstrates the selective quantification of a small protein at low nmol/L concentrations. The approach has future application in the bioanalysis of small protein therapeutics or small protein biomarkers particularly where immunoassay methods do not provide adequate selectivity, or where antibodies can not be raised.

There has been a recent increase in the number of peptide therapeutics requiring quantitative LC-MS/MS bioanalytical assays. A number of papers have reviewed the issues encountered in developing bioanalytical
assays for peptides [1-3]. The problems include adsorption, biological instability, difficult chromatography, low MS/MS response and protein or antibody binding. For peptides these challenges can often be overcome,
however, for small proteins (>10000 Mwt) other approaches need to be considered. Small proteins demonstrate multiple charging under electrospray ionisation conditions with the formation of many multiply charged ions. Individual ions can be used for quantification, however, charge distribution means that the overall sensitivity is greatly reduced [4].

Additionally, the ratio of charge states can change depending on a number of factors, resulting in problems when monitoring only single ions [1]. Monitoring and summing the data for several ionisation states can increase response, but the endogenous background gains means that improvements to the signal to noise ratio are often negligible. Furthermore, larger molecules demonstrate a wide isotopic distribution which complicates the decision as to which ions to quantify, and limits the sensitivity further.

Free to read

This article has been unlocked and is ready to read.


Digital Edition

Chromatography Today - Buyers' Guide 2022

October 2023

In This Edition Modern & Practical Applications - Accelerating ADC Development with Mass Spectrometry - Implementing High-Resolution Ion Mobility into Peptide Mapping Workflows Chromatogr...

View all digital editions


NGVS 2024

May 23 2024 Beijing, China


May 28 2024 Tel Aviv, Israel


May 28 2024 Leuven, Belgium

PREP 2024

May 28 2024 Philadelphia, PA, USA


May 29 2024 Beijing, China

View all events