Investigating effects of sample pretreatment on protein stability using size‐exclusion chromatography and high‐resolution continuum source atomic absorption spectrometry

Abstract

In this study, size-exclusion chromatography and high-resolution atomic absorption spectrometry methods have been developed and evaluated to test the stability of proteins during sample pretreatment. This especially includes different storage conditions but also adsorption before or even during the chromatographic process. For the development of the size exclusion method, a Biosep S3000 5 μm column was used for investigating a series of representative model proteins, namely bovine serum albumin, ovalbumin, monoclonal immunoglobulin G antibody, and myoglobin. Ambient temperature storage was found to be harmful to all model proteins, whereas short-term storage up to 14 days could be done in an ordinary refrigerator. Freezing the protein solutions was always complicated and had to be evaluated for each protein in the corresponding solvent. To keep the proteins in their native state a gentle freezing temperature should be chosen, hence liquid nitrogen should be avoided. Furthermore, a high-resolution continuum source atomic absorption spectrometry method was developed to observe the adsorption of proteins on container material and chromatographic columns. Adsorption to any container led to a sample loss and lowered the recovery rates. During the pretreatment and high-performance size-exclusion chromatography, adsorption caused sample losses of up to 33%.

A novel immunoassay for quantitative drug abuse screening in serum

“…Long-term storage and sample handling could affect the stability of serum compounds (ElMaouche et al, 2016;Rakow et al, 2014;Setoyama et al, 2015). Therefore, it is possible that various metabolites or degradation products were present, which was not observed within the measurements.…”

A design of experiments concept for the minimization of nonspecific peptide adsorption in the mass spectrometric determination of substance P and related hemokinin‐1

“…However, LC-MS/MS applications were not described for the determination of HK-1 or lacked the sensitivity to determine endogenous blood levels of SP [11]. One of the principal reasons for the lack of sensitivity and a bad reproducibility by measuring biomolecules in LC-MS/MS applications are adsorption processes to the container material and components of the analytical system [12,13]. As SP and HK-1 are positively charged and moderately hydrophobic peptides (Table 1), they unify the potential to adsorb to glass as well as plastic surfaces [14][15][16].…”

Determination of protein charge variants with (imaged) capillary isoelectric focusing and capillary zone electrophoresis

“…One of the main concerns is the sample stability during the preparation procedure. Rakow examined changes of protein stability after different kinds of sample pretreatment, namely filtration, freeze‐thaw cycles, and ultrasonic bath . Especially filtration reduces the protein concentration due to adsorption effects.…”