Cynthia Williams; Anne Fenton; Lauren Nagy; Qiuting Hong; L. Renee Ruhaak; Satya Dandekar; Carlito Lebrilla
UC Davis, Davis, CA
Monitoring the degree of glycosylation with the absolute protein quantitation in plasma of HIV patients.
The immune system is highly dysregulated in patients with HIV infection. Current studies aim to understand this process to allow for better prevention, earlier diagnosis and a cure. Immunoglobulin concentrations are abnormal in HIV patients and they display altered glycosylation patterns. However, absolute protein quantitation, simultaneously, with site-specific glycosylation analysis of human plasma in HIV samples has previously not been achieved. We are developing methods for quantitation of glycosylated proteins on the protein-specific and site-specific level. In this study, we used multiple reaction monitoring (MRM) to quantitate immunoglobulins A, G, and M (IgA, IgG, and IgM) proteins and their site-specific glycans to examine whether immunoglobulin-specific glycosylation varies in HIV infection.
Plasma samples from 26 male patients and 11 controls were collected and HIV infected patients were broken into 3 groups: No Therapy (n=11), Therapy (n=11), and Long Term Non Progressors (n=4). Two uL of patient sera and IgA, IgG, and IgM standards were digested using trypsin after reduction and alkylation. Protein and glycopeptide quantitation was performed using a QqQ MS in dynamic MRM mode coupled with a UPLC system. Unglycosylated peptides were used for absolute protein quantitation. Site-specific glycosylation was normalized to absolute protein abundances to determine degree of glycosylation.
Presented here is a high-throughput MRM method for absolute quantitation of IgG, IgA and IgM and their glycosylation analysis directly from 2 uL of serum. A 10 &11-minute UPLC gradient was used with a C18 stationary phase and were employed in the development of the method to quantify protein and site-specific glycan abundances for IgG and IgA & IgM. IgG, A, and M concentrations, as well as subclasses and glycosylation patterns were monitored through 14 MRM transitions for peptides and 53 for glycopeptides.
Our rapid throughput platform was applied to HIV samples (26 patients and 11 controls). For IgG, the peptide common from each subclass was used to quantify the total IgG content. Because this peptide is common to all four subclasses, it correlates linearly to the total IgG protein abundances. Average IgG concentrations were 10.1, 12.3, 13.7, and 19.2 mg/mL in HIV Negative (Neg), Therapy (Tx), Long Term Non-Progessors (LTNP), and No Therapy (No Tx) cases, respectively. Differences with statistical significance were observed in the protein concentrations and in specific IgG glycoforms as well. Galactose deficient glycopeptides were observed to be significantly higher in the HIV positive cases. In IgA, there were no differences observed on the protein level, but on the glycosylation level, Hex4HexNAc4NeuAc1 was shown to be significantly lower in the Tx groups. In comparison to the IgG analysis, the IgM protein concentration levels were only elevated in the No Tx cases. Interestingly, although there were only 4 patient samples within the LTNP group, a positive association was observed between levels of high mannose type glycans and the LTNP response. These results show that the immune responses of HIV patients are clearly altered and that glycosylation is a very important factor.