In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15. s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.

Based on the previous demonstration of surface (S-) layer protein glycosylation in Lactobacillus buchneri 41021/251 and because of general advantages of lactic acid bacteria for applied research, protein glycosylation in this bacterial species was investigated in detail. The cell surface of L. buchneri CD034 is completely covered with an oblique 2D crystalline array (lattice parameters, a = 5.9 nm; b = 6.2 nm; γ ∼77) formed by self-assembly of the S-layer protein SlpB. Biochemical and mass spectrometric analyses revealed that SlpB is the most abundant protein and that it is O-glycosylated at four serine residues within the sequence S152-A- S154-S155-A-S157 with, on average, seven Glc(α1-6) residues, each. Subcellular fractionation of strain CD034 indicated a sequential order of SlpB export and glucosylation as evidenced by lack of glucosylation of cytosolic SlpB. Protein glycosylation analysis was extended to strain L. buchneri NRRL B-30929 where an analogous glucosylation scenario could be detected, with the S-layer glycoprotein SlpN containing an O-glycosylation motif identical to that of SlpB. This corroborates previous data on S-layer protein glucosylation of strain 41021/251 and let us propose a species-wide S-layer protein O-glucosylation in L. buchneri targeted at the sequence motif S-A-S-S-A-S. Search of the L. buchneri genomes for the said glucosylation motif revealed one further ORF, encoding the putative glycosylâ€hydrolase LbGH25B and LbGH25N in L. buchneri CD034 and NRRL B-30929, respectively, for which we have indications of a glycosylation comparable to that of the S-layer proteins. These findings demonstrate the presence of a distinct protein O-glucosylation system in Gram-positive and beneficial microbes.

While it is now evident that the two Bacteroidales species Bacteroides fragilis and Tannerella forsythia both have general O-glycosylation systems and share a common glycosylation sequon, the ability of these organisms to glycosylate a protein native to the other organism has not yet been demonstrated. Here, we report on the glycosylation of heterologous proteins between these two organisms. Using genetic tools previously developed for Bacteroides species, two B. fragilis model glycoproteins were expressed in the fastidious anaerobe T. forsythia and the attachment of the known T. forsythia O-glycan to these proteins was demonstrated by liquid chromatography electrospray ionization tandem mass spectrometry. Likewise, two predominant T. forsythia glycoproteins were expressed in B. fragilis and glycosylation with the B. fragilis O-glycan was confirmed. Purification of these proteins from B. fragilis allowed the preliminary characterization of the previously uncharacterized B. fragilis protein O-glycan. Based on mass spectrometric data, we show that the B. fragilis protein O-glycan is an oligosaccharide composed of nine sugar units. Compositional and structural similarities with the T. forsythia O-glycan suggest commonalities in their biosynthesis. These data demonstrate the feasibility of exploiting these organisms for the design of novel glycoproteins.

Alpha-l-Iduronidase(IDUA) was the first enzyme replacement therapy approved for mucopolysaccharidosis type I and the corresponding recombinant protein drug, Aldurazyme®, is commercially available. In the frame of gel-based mass spectrometrical characterization of protein drugs, we intended to identify protein sequence and possible protein modifications. Moreover, we were interested in which aggregation/complex form Aldurazyme® would exist, which complexes were enzymatically active and in which form the naturally occurring enzyme would be present in the brain. Aldurazyme® was run on 2DE gel electrophoresis, spots were excised, in-gel digested with several proteases and identified by nano-LC-ESI-MS/MS on an ion trap. IDUA-activity was determined by a fluorometric principle. Blue-native gel electrophoresis with subsequent immunoblotting was carried out to show the presence of protein complexes.The protein was unambiguously identified by 100% sequence coverage; several amino acid substitutions were detected and protein modifications were novel phosphorylations on S59 and S482, histidine methylation at H572 and provide evidence for already known N-glycosylations. Four Aldurazyme® complexes that all were enzymatically active, were observed while a single complex was observed for the physiologically occurring IDUA in the brain.The findings are relevant for understanding chemistry, physiology, pharmacology and medicine of IDUA, design of further and interpretation of previous work.