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Access Agilent eNewsletter January 2016

Fast, accurate absolute-quantification of proteins and antibodies with Agilent 8800 ICP-QQQ

Philippe De Raeve
Scientific Director, Quality Assistance Contract Research Organisation, Belgium

Amir Liba
Agilent US SPSD Application Chemist Team Manager

In life science research, analytical methods that allow for accurate quantification of proteins and peptides, without the need for a compound-specific standard, are highly beneficial, as many compounds are unknowns.

Today there is a novel technique for protein quantification that utilizes inductively coupled plasma mass spectrometry (ICP-MS) to determine protein concentration via the signal response for a heteroelement contained within the protein. Triple quadrupole ICP-MS (ICP-QQQ) is capable of operating in MS/MS mode to effectively remove spectral overlaps. This allows access to multiple isotopes of many elements, which enables isotope dilution mass spectrometry (IDMS) analysis. IDMS quantifies the target compound based on the heteroelement isotope ratio measured in a mixture of the sample and a spike of a certified standard of known isotopic composition. The concentration measurement in each sample is made independently (without reference to an external standard) via the isotope ratio, so IDMS can be used to quantify a compound without the need for compound specific calibration standards, and so allows accurate quantification of unknown proteins and peptides.

Sulfur isotope ratio analysis with Agilent solution

Most proteins contain sulfur from methionine and cysteine residues. Sulfur determination is challenging for conventional quadrupole ICP-MS due to the element’s high ionization potential (10.4 eV) that leads to low sensitivity, and the occurrence of multiple polyatomic ions that give rise to spectral overlaps on all isotopes of S, especially in an organic matrix. High resolution ICP-MS (HR-ICP-MS) can be used for the analysis of S, but, apart from the much higher cost of the instrumentation, it suffers a 10-fold reduction in ion transmission efficiency (sensitivity) at the mass resolution required to separate the S isotopes from the spectral interferences.

Employing the MS/MS capabilities of the Agilent 8800 ICP-QQQ, we developed a high-accuracy IDMS method to quantify sulfur via the measurement of the 32S and 34S isotopes. A mass-shift mode was used, where the analyte ions are reacted with O2 collision/reaction cell gas, and the isotope-specific SO+ product ions are measured at M + 16 amu, that is m/z 48 and 50.

Resolve sensitivity differences using high accuracy double spike IDMS technique

Since the enriched isotope spike solution (H234SO4) was prepared by oxidation of a solid 34S powder, the true concentration of 34S in the spiking solution had to be determined by reverse IDMS, using a double-spiking method. A National Institute of Standards and Technology (NIST) certified solution of SO4 with natural sulfur isotopic abundance was used as the reference standard to quantify the 34S in the spiking solution. Double IDMS also compensates for any sensitivity differences between 32S and 34S—i.e. mass bias correction is automatically applied.

Full method verification achieved using ICP-QQQ

For our analysis, Bovine Serum Albumin (BSA) was selected as a model compound to test the method. BSA has the formula C2932H4614N780O898S39 and is therefore 1.833% S; the NIST certified standard reference material (SRM) 927e BSA (7% solution) contains 67.38 ± 1.38 g/L (± 2.0 %) of S. Several separate weighings (per the levels shown in Table 1) of the BSA SRM were digested and measured. Each solution was analyzed using ICP-QQQ, and the sulfur concentration was determined using IDMS. The results shown in Table 1 demonstrate excellent accuracy and precision, with recoveries of nearly 100% and RSD less than 0.35%.

Actual weight of NIST 927e BSA solution (mg) Nominal mass of BSA (mg, approx.) Sulfur (µg, approx) Sulfur (µg/g, approx) Average recovery (%) RSD (%) n
26.05; 24.67; 23.37 1.6 30 0.6 101.29 0.34 3
30.93; 32.09; 31.34 2.1 40 0.8 101.30 0.21 3
40.33; 40.19; 39.72;
39.60; 40.01; 40.30
2.6 50 1.0 101.26 0.22 6
47.71; 48.30; 48.12 3.2 60 1.2 101.17 0.13 3
55.56; 55.72; 56.03 3.7 70 1.4 101.17 0.12 3

Table 1. Analysis of different sample digests of NIST 927e BSA SRM solution

The analytical method was also evaluated in terms of:

  • Matrix effects from potential formulation buffer ingredients. Results were consistent in the presence of various individual components and mixed formulation buffer.
  • Accuracy in the presence of non-protein sulfur:
    • Sulfate. A NIST BSA 927e solution was spiked with SO4 (~5% of BSA sulfur). Part of this solution was filtered through a 3 KDa molecular weight cut-off membrane to separate any possible low molecular weight sulfur-containing contaminants. The corrected BSA recovery in the spiked solution was 99.6 %, indicating no contribution from the sulfate.
    • Methionine. Methionine was added to solutions of NIST BSA 927e and human immunoglobulin (IgG). The solutions were filtered and methionine was measured in the original solutions and in the filtrate. The results given in Table 2 show excellent recovery for methionine in the filtrate at 99.1 and 99.5% respectively. This clearly indicates that any possible contamination of low molecular weight sulfur-containing molecules can be selectively removed through filtration.
NIST BSA solution IgG solution
Methionine added (mM) 2.05 2.05
Methionine in filtrate (mM) 2.03 2.04
% Recovery 99.1 99.5

Table 2. Recovery of methionine

Application to the analysis of trastuzumab solution

Trastuzumab is a monoclonal antibody that interferes with the human epidermal growth factor receptor 2 (HER2/neu receptor). In this application, the IDMS-ICP-QQQ method was used to measure the concentration of sulfur in a solution containing trastuzumab. Good agreement with the expected value was achieved as shown in Table 3.

Expected concentration Measured concentration %RSD (n=3) % Recovery
Sulfur 21 mg/mL 20.53 mg/g 0.02 97.8

Table 3. Analysis of trastuzumab solution

Accurate quantification of biological molecules with Agilent 8800 ICP-QQQ

The Agilent 8800 ICP-QQQ with MS/MS mode provides high sensitivity and effective interference reduction for multiple sulfur isotopes, allowing determination of sulfur by IDMS, using the 34S/32S ratio. This allows the accurate quantification of biological molecules such as proteins, peptides, and antibodies, via their sulfur heteroelement content. For more details of this study download Application Note 5991-6118EN. To learn more about this collaboration and Quality Assistance S.A., visit

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