Sialic Acid Speciation Using Capillary Electrophoresis: Optimization of Analyte Derivatization and Separation

Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) was employed for rapid sialic acid speciation, facilitating the quantitative determination of <i>N</i>-glycolylneuraminic acid (Neu5Gc) and <i>N</i>-acetylneuraminic acid (Neu5Ac) on glycoproteins. Derivatization of the sialic acids with 2-aminoacridone (2-AMAC), using classical reductive amination in a nonaqueous solvent, led to the spontaneous decarboxylation of the sialic acid residues as determined by CE-LIF and offline mass spectrometric analysis. Modification of both the labeling conditions, to drive the decarboxylation reaction to completion and the CE-LIF parameters to separate the neutral species by complexation with a neutral coated capillary and borate reversed polarity, led to a robust platform for the rapid, sensitive, and quantitative speciation of sialic acids. The method can readily be used for quality control of recombinant biopharmaceuticals

The positions of the mutated amino acids in the structure of PCNA.

<p>(A) Bar diagram of yeast PCNA. The positions of the altered amino acids, and of the IDCL are indicated. (B) The positions of the mutated amino acids at the subunit interface are shown in different colors in a schematic ribbon diagram representing the three dimensional structure of the PCNA trimer (source: Krishna, T.S.R at al. from RCSB PDB, PDB ID:1PLR) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161307#pone.0161307.ref007" target="_blank">7</a>]. The monomers are depicted in different colors.</p

In the II99,100AA PCNA mutant strain the <i>REV3</i> branch of translesion synthesis is inactivated.

<p>(A-B) The II99,100AA PCNA mutant shows epistasis with <i>RAD18</i> upon UV treatment. (C-D) The II99,100AA PCNA mutant affects the Rev3 pathway. On (A) and (C) ten fold serial dilutions of cells with the indicated genotypes were spotted on plates and exposed to UV doses indicated on the right. (B) and (D) show quantitative analysis of UV-survival of the indicated strains. (E) UV-induced mutagenesis is abolished in the II99,100AA PCNA mutant strain. Forward mutation rates at the <i>CAN1</i> locus were determined after exposing the cells to the indicated UV doses. (B), (D), and (E) represent the average of three experiments. Standard deviations are indicated. P-values representing the significance of difference are also shown on (E). *:p<0.05 ** p<0.01, *** p<0.001 ns: no statistical difference.</p

Percentage of cells displaying the large budded (dumbbell) morphology of yeast strains expressing the PCNA variants.

<p>Percentage of cells displaying the large budded (dumbbell) morphology of yeast strains expressing the PCNA variants.</p

Phenotypic characterization of the mutants.

<p>(A) Growth of the strains at different temperatures. Approximately 100 cells of each strain were plated on rich medium and the plates were incubated at the indicated temperatures for 2 days. (B) Sensitivity of the strains to HU. Ten fold serial dilutions of the indicated strains were spotted on YPD plates containing the given amount of HU. (C) Sensitivity of the strains to DNA damaging agents. Cells were spotted as described for (B) on plates either exposed to the indicated UV dose, or containing the indicated amount of MMS. For (B) and (C) several HU, UV, and MMS doses were applied, but only plates with the most appropriate doses are presented. (D) Quantitative assays of UV and MMS induced killing of the indicated strains. The results represent the average of three experiments. Standard deviations are indicated.</p

Spontaneous mutation rates in yeast strains expressing the PCNA variants.

<p>Spontaneous mutation rates in yeast strains expressing the PCNA variants.</p

The II181,182AA PCNA behaves like wild type in its interaction with Rad54 <i>in vitro</i>.

<p>GST-Rad54 (3 μg) bound to glutathione-Sepharose beads was incubated with purified wild type (left panel), or II181,182AA mutant PCNA (right panel) (5–5 μg). After washing bound proteins were eluted with glutathione. Aliquots of each sample, taken from the input (I), from the unbound fraction (U), from the last wash (W), and from the glutathione-eluted proteins (E), were analyzed on 10% SDS polyacrylamide gel.</p

Complex formation between PCNA and Rev1.

<p>(A) GST-pull-down assay with Rev1-PAD and wild type, or II99,100AA PCNA. GST-Rev1-PAD (5 μg) immobilized on glutathione-Sepharose beads was incubated with purified wild type (left panel), or II99,100AA mutant PCNA (right panel) (5–5 μg). After washing bound proteins were eluted with glutathione. Aliquots of each sample, taken from the input (I), from the unbound fraction (U), from the last wash (W), and from the glutathione-eluted proteins (E), were analyzed on 10% SDS polyacrylamide gel. (B) GST-pull-down with Rev1 (5 μg) and wild type, or II99,100AA mutant PCNA (3–3 μg). Experiments were carried out as described for (A), but GST-Rev1 was used instead of GST-Rev1-PAD, and elution was achieved by PreScission protease cleavage of Rev1 from GST. The position of the cleaved Rev1 without the GST tag in the elution fraction is also marked.</p

The II181,182AA PCNA mutations affect HR differently based on the nature of the treatment of the cells.

<p>(A) The II181,182AA PCNA mutant is epistatic to both <i>RAD52</i> and <i>RAD54</i> upon UV-treatment. (B) Quantitative analysis of UV-survival of the <i>rad52 pol30-II181</i>,<i>182AA</i> double mutant. (C) X-ray (D) bleomycin (E) HU sensitivity of the <i>rad52 pol30-II181</i>,<i>182AA</i> double mutant strain. In panels (A), (C), (D), and (E) ten fold serial dilutions of strains with the indicated genotypes were spotted on YPD plates containing the given amount of bleomycin, or HU, or irradiated with the indicated UV or X-ray doses.</p

High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the N‑Glycome of a Recombinant Human Erythropoietin

Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS² based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due to their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry