An LC/MS-based method is established for the differentiation and authentication of specimens and commercial samples of Panax ginseng (Oriental ginseng) and Panax quinquefolius (American ginseng). This method is based on the separation of ginsenosides present in the ginseng methanolic extracts using high-performance liquid chromatography (HPLC), followed by detection with electrospray mass spectrometry. Differentiation of ginsenosides is achieved through simultaneous detection of intact ginsenoside molecular ions and the ions of their characteristic thermal degradation products. An important parameter used for differentiating P. ginseng and P. quinquefolius is the presence of ginsenoside Rf and 24-(R)-pseudoginsenoside F11 in the RICs of Oriental and American ginsengs, respectively. It is important to stress that ginsenoside Rf and 24(R)-pseudoginsenoside F11, which possess the same molecular weight and were found to have similar retention times under most LC conditions, can be unambiguously distinguished in the present HPLC/MS method. The method developed is robust, reliable, reproducible, and highly sensitive down to the nanogram level.

Objective. The programmed death 1 (PD-1) molecule is a negative regulator of T cells, and a genetic association between PD-1 and systemic lupus erythematosus and rheumatoid arthritis (RA) in Caucasians has been reported. The aim of this study was to investigate the association of PDCD1 polymorphisms and haplotypes with RA in the Chinese population.Methods. Three single-nucleotide polymorphisms (SNPs), PD-1.1 G/A, PD-1.3 G/A, and PD-1.5 C/T, were genotyped in 180 patients with RA and 647 healthy controls in a case-control association study. Analyses of the association of genotypes and alleles with disease, haplotype construction, and linkage disequilibrium (LD) were performed.Results. We constructed haplotypes with the alleles of markers PD-1.1 G/A and PD-1.5 C/T and found that the GT haplotype was overrepresented in patients with RA (31%) compared with controls (23%) (P ‫؍‬ 0.001, odds ratio [OR] 1.54, 95% confidence interval [95% CI] 1.18-1.99). Among GT double homozygotes the risk of RA was increased even further (OR 2.31, 95% CI 1.31-4.08, P ‫؍‬ 0.006). We also observed that the AA genotype of SNP PD-1.1 was associated with a decreased risk for developing RA (OR 0.38, 95% CI 0.15-0.99, P ‫؍‬ 0.034). No association for SNP PD-1.5 in RA was found, and SNP PD-1.3 was nonpolymorphic in the Chinese population.Conclusion. Our results support the involvement of PDCD1 as a susceptibility gene for RA in the Chinese population.

Disulfide bonds formed between cysteine residues stabilize the native structure of many proteins.[1] These covalent crosslinkages are part of the tertiary structure and introduce conformational constraints to the polypeptide architecture, thus improving the thermodynamic stability. Mass spectrometry has become increasingly important in the characterization and identification of proteins as a result of its capability for highly specific measurements on relatively small amounts of analyte. Protein characterization and identification relies on database searches using primary sequence information obtained by tandem mass spectrometry (MS n or MS/MS). The top-down approach [2] applied to intact proteins ions is an efficient MS/MS method which is potentially suited for the high-throughput characterization of proteins. The problems addressed by the top-down approach include verification of the protein sequence, localization of errors in DNA-predicted sequences, de novo sequencing, as well as the analysis of pre-or posttranslational modifications (PTMs). However, such PTMs as disulfide bonds still remain a challenge for traditional MS/MS techniques that utilize vibrational excitation (VE), as they break peptide bonds preferentially in protonated polypeptides and leave the disulfide bonds intact.[3] As a result, information on the primary sequence is limited to parts of the protein that is not constrained by disulfide bonds. This restriction complicates primary sequence elucidation and limits the utility of automated database searches. The problem of disulfide bonds is solved conventionally by their reduction and alkylation [4] prior to MS/MS measurements. However, the additional step involving solution-phase chemistry, including subsequent purification, greatly increases the analysis time and the risk of sample loss. Another approach is to use an alternative iondissociation strategy. Chrisman and McLuckey have shown that VE of deprotonated polypeptides induced by collisionally activated dissociation (CAD) [5] breaks disulfide bonds with high efficiency.[6] However, CAD of peptide anions often produces complicated mass spectra that, together with backbone cleavages, contain significant amounts of neutral mass losses and rearrangement products which make such MS/MS spectra of limited value for sequencing. Electron-capture dissociation (ECD) [7] stands alone in its ability to cleave SÀS bonds preferentially in peptide polycations (Scheme 1).[3] A drawback of this technique is that the capture of a low-energy electron by multiply charged polypeptides results in charge reduction prior to bond cleavage, which in turn leads to a charge decrease in one of the two fragments. As species need to contain at least one charge to be detectable in mass spectrometry, the charge reduction in ECD can lead to a loss of information. Previously, when magnetic sector instruments were widely used, high-energy CAD (HE CAD) was utilized to produce cleavage of numerous disulfide bonds in peptides.[8] The abundance of this fragmentation channel comp...

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using c + 57 or z• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.