Letter: Collision energy and cone voltage optimisation for glycopeptide analysis.

Instrument tuning commonly used for peptide analysis and for proteomics causes a high degree of fragmentation for glycopeptides. This results in a strongly biased glycosylation pattern. To obtain correct results for glycopeptides, both the cone voltage and the collision energy has to be reduced significantly. A suitable standard for tuning the instrument for glycopeptide analysis is aspartic acid (which fragments under similar conditions as glycopeptides); while low mass sugar fragments (for example, at 657.3 Da) are good indicators for the presence/absence of glycopeptide fragmentation.

Investigation of genetic variants of alpha-1 acid glycoprotein by ultra-performance liquid chromatography-mass spectrometry.

Genetic variants of human plasma alpha-1 acid glycoprotein (AGP) have been studied in cancer, compared with a group of healthy control. AGP has four genetic variants: AGP F1, F2, and S variants correspond to the ORM1 gene whereas AGP A corresponds to the ORM2 gene. The proportion of ORM1 and ORM2 variants were studied in plasma using a novel UPLC-MS method. Plasma total AGP level was 0.5 +/- 0.2 g L(-1) and the proportions of the ORM1 and ORM2 variants were 76.3 +/- 8.2% and 23.7 +/- 8.2%, respectively. In cancer plasma AGP levels increased fourfold and the proportion of ORM1 variants increased to 88.7 +/- 6.8%. Changes in the proportion of genetic variants due to cancer were clearly significant, as shown by statistical analysis. Three different cancer types have been studied, lymphoma, melanoma, and ovarian cancer. The results did not show any difference depending on cancer type. The results indicate that, in accordance with prior expectations, the ORM1 variant is predominantly responsible for the acute-phase property of AGP.

GlycoMiner: a new software tool to elucidate glycopeptide composition.

New computer software, GlycoMiner, has been developed to automatically identify tandem (MS/MS) spectra obtained in liquid chromatography/mass spectrometry (LC/MS) runs which correspond to N-glycopeptides. The program complements conventional proteomics analysis, and can be used in a high-throughput environment. The program interprets the spectra and determines the structure of the corresponding glycopeptides. GlycoMiner runs under Windows, can process spectra obtained on various instruments, and can be downloaded from our website (w3.chemres.hu/ms/glycominer). The algorithm works similarly to a human expert; evaluates the low mass oxonium ions; deduces oligosaccharide losses from the protonated molecule; and identifies the mass of the peptide residue. The program has been tested on tryptic digests of two glycopeptides: AGP (which has five different N-glycosylation sites) and transferrin (with two N-glycosylation sites). Results have been evaluated both manually and by GlycoMiner. Out of 3132 MS/MS spectra 338 were found to correspond to glycopeptides; identification by GlycoMiner showed a 0.1% false positive and 0.1% false negative rate. From these it was possible to identify 196 glycan structures manually; GlycoMiner correctly identified all of these, with no false positives. The rest were low quality spectra, not suitable for structure assignment.

Lipophilicity of aminopyridazinone regioisomers.

Ten pairs of pyridazinone regioisomers were prepared, and their lipophilicity was described by the logarithm of the octanol/water partition coefficient (log P) determined experimentally and calculated with prediction methods. The 4- and 5-(substituted amino)-3(2H)-pyridazinone regioisomers were synthesized by nucleophilic substitution of one of the chloro atoms of 4,5-dichloro-2-methyl-3(2H)-pyridazinone or its 6-nitro derivative. Structures of new compounds were proven by spectroscopic methods. The experimental log P values were obtained by a shake flask method in octanol and a Sörensen buffer (pH 7.4) solvent system. A consequent difference was found in the lipophilicity of regioisomers. For each isomer pair, the log P value of the 4-isomer was significantly (average by 0.75 log unit) higher than that of the 5-isomer. Some quantum chemical calculations as well as X-ray analysis of two pairs of regioisomers were also carried out to gain insight into the structural differences of regioisomers. The log P values were calculated by the fragmental approach KOWWIN and a QSPR analysis (3DNET). The a priori KOWWIN gave poor agreement, but with the programs KOWWIN with EVA (experimental value adjusted) and 3DNET, the results were generally in agreement with experiment.