Simple correction improving long-term reproducibility of HPLC-MS

Summary Signal intensities in long series of HPLC-MS experiments often vary, which decrease reproducibility and may cause bias in the results. It was found that the sensitivity of various components change differently; in our case variability is in the order of 20-40%; and it is most likely due to changing conditions in ESI ionization. The most often used intensity correction methods do not take this effect into account. The change in signal intensities (peak areas) can be well described by a polynomial function; we found that a 4th order polynomial is most often suitable. We suggest a simple correction algorithm based on polynomial fitting. When the experiments were inherently well reproducible, this correction improved reproducibility from 12% to 3% (on average for various components). When random errors were larger, this improvement was less significant (15% to 12% in nano-ESI), but nevertheless essential in order to avoid possible bias in the results

HPLC enrichment/isolation of proteins for post-translational modification studies from complex mixtures

The paper describes a macroporous RP-HPLC method for separation and isolation/enrichment of proteins from complex mixtures. The method is robust and efficient; using 2.1 or 4.6 mm diameter columns provides sufficient material for subsequent proteomic analysis. The main advantage of the method is that most protein variants are isolated in the same fraction, as separation is not based on differences in isoelectric point. This is highly advantageous for studying complex mixtures and post-translational modifications. Examples related to glycosylation analysis are discussed in detail

Changes of protein glycosylation in the course of radiotherapy

This is the first study of changes in protein glycosylation due to exposure of human subjects to ionizing radiation. Site specific glycosylation patterns of 7 major plasma proteins were analyzed; 171 glycoforms were identified; and the abundance of 99 of these was followed in the course of cancer radiotherapy in 10 individual patients. It was found that glycosylation of plasma proteins does change in response to partial body irradiation (~60 Gy), and the effects last during follow-up; the abundance of some glycoforms changed more than twofold. Both the degree of changes and their time-evolution showed large inter-individual variability

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP : Phosphocholine Cytidylyltransferase

Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40 degrees C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line

Mass spectrometry-based analysis of macromolecular complexes of Staphylococcus aureus uracil-DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA. The uracil base is among the most frequently occurring erroneous bases in DNA, and is cut out from the phosphodiester backbone via the catalytic action of uracil-DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil-DNA glycosylase. Interestingly, both uracil-DNA glycosylase (Staphylococcusaureus uracil-DNA glycosylase; SAUDG) and its inhibitor (S.aureus uracil-DNA glycosylase inhibitor; SAUGI) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil-DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI. Here, we investigated whether these mutations drastically perturb the interaction with SAUDG. To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these K-d values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil-DNA glycosylase inhibitor proteins

Simple correction improving long-term reproducibility of HPLC-MS

Chromatographic peak areas in long series of high-performance liquid chromatography-MS experiments often vary, which decrease reproducibility and may cause bias in the results. It was found that the sensitivity of various components change differently; in our case, variability is in the order of 20-40%, and it is most likely due to changing conditions in electrospray ionization (ESI). The most often used peak area correction methods do not take this effect into account. The change in peak areas can be well described by a polynomial function; we found that a fourth-order polynomial is most often suitable. We suggest a simple correction algorithm based on polynomial fitting. When the experiments were inherently well reproducible, this correction improved reproducibility from 12% to 3% (on average for various components). When random errors were larger, this improvement was less significant (15% to 12% in nano-ESI) but nevertheless essential in order to avoid possible bias in the results. Copyright © 2015 John Wiley & Sons, Ltd

Quantitative comparison of tandem mass spectra obtained on various instruments

The similarity between two tandem mass spectra, which were measured on different instruments, was compared quantitatively using the similarity index (SI); defined as the dot product of the square root of peak intensities in the respective spectra. This function was found to be useful for comparing energy dependent tandem mass spectra obtained on various instruments. Spectral comparisons show the similarity index in a 2D “heat map”; indicating which collision energy combinations result in similar spectra; and how good this agreement is. The results and methodology can be used in the pharma industry to design experiments and equipment well suited for good reproducibility. We suggest that to get good long term reproducibility, it is best to adjust the collision energy to yield a spectrum very similar to a reference spectrum. It is likely to yield better results than using the same tuning file, which e.g. does not take into account that contamination of the ion source due to extended use may influence instrument tuning. The methodology may be used to characterize energy dependence on various instrument types; to optimize instrumentation; and to study the influence or correlation between various experimental parameters