Hb Kirikiriroa [α57(E6)Gly→Cys; HBA1: c.172G>T]: A Novel Unstable α-Globin Variant with Oxidized Derivatives Interfering with Hb A1c.

We report the identification of a novel hemoglobin (Hb) variant [α57(E6)Gly→Cys; HBA1: c.172G>T], to be referred to as Hb Kirikiriroa. The variant was detected in five subjects from two families, with familial relationship established between the families following diagnosis. A persistently elevated Hb A1c over a 1-year period prompted hemoglobinopathy screening in an adolescent male of New Zealand (NZ) European descent (case 1). Capillary electrophoresis (CE) revealed the variant was negatively charged and susceptible to oxidation, with multiple abnormal peaks detected (0.4-5.1% total Hb). Hb A1c analysis by cation exchange high performance liquid chromatography (HPLC) was the first indication of the variant in a pregnant female of NZ European descent (case 2). Cases 1 and 2 had normal complete blood counts. Isopropanol stability testing provided evidence the variant was unstable. We herein describe the characterization of Hb Kirikiriroa and clinical significance of the variant for interference with Hb A1c analysis by CE and cation exchange HPLC.

Immuno-MALDI-MS for Accurate Quantitation of Targeted Peptides from Volume-Restricted Samples.

The immuno-MALDI-MS method can be used to quantify low-abundance proteins from clinical samples that offer only a limited amount of material for analysis. An internal standard, in the form of a stable isotope-labeled peptide, is used to ensure reproducible and absolute quantitation. The protocol described here was optimized for the quantitation of AKT1 and AKT2, but we offer instructions on how to adapt the method to target other proteins. The described workflow is compatible with automation via a liquid handling robot for high-throughput applications.

Hb Tacoma: G>T or G>C, and Does It Matter?

Hb Tacoma [ß30(B12)Arg→Ser] is a missense variant that is caused by either an AGG>AGT or AGG>AGC substitution at codon 30 of the HBB gene. Currently, the latter is classified as a rare cause of ß0-thalassemia (ß0-thal). We propose that HBB: c.93G>C has been incorrectly assigned as ß0-thal and discuss whether HBB: c.93G>T or HBB: c.93G>C should be classified as ß+-thal instead, or as ß-globin variants without thalassemic effect. We present several subjects who are heterozygous for Hb Tacoma, one with HBB: c.93G>T and two with HBB: c.93G>C, to support our conclusions.

Use of MALDI-TOF MS to Discriminate between Biofilm-Producer and Non-Producer Strains of Staphylococcus epidermidis.

For the management of Staphylococci coagulase-negative infection, often related to biofilm formation, rapid and accurate identification is necessary in choosing a correct antibiotic therapy. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is becoming increasingly important for bacterial identification over traditional methods. Our aim was to validate the use of MALDI to discriminate Staphylococcus epidermidis biofilm-producing strains. Clinical strains coming from suture wires were identified and their protein profiles were compared to that obtained from two ATCC reference strains (biofilm producer and non-producer). MALDI identified the eighteen isolates as S. epidermidis, combining sixteen profiles with the biofilm producer and two with the non-producer, confirming the results of crystal violet assay. Our data highlight that MALDI can be considered a good tool to discriminate between biofilm-producer and non-producer strains of S. epidermidis, thus helping to establish an effective antibiotic therapy.

Whole-Cell MALDI-TOF MS Versus 16S rRNA Gene Analysis for Identification and Dereplication of Recurrent Bacterial Isolates.

Many ecological experiments are based on the extraction and downstream analyses of microorganisms from different environmental samples. Due to its high throughput, cost-effectiveness and rapid performance, Matrix Assisted Laser Desorption/Ionization Mass Spectrometry with Time-of-Flight detector (MALDI-TOF MS), which has been proposed as a promising tool for bacterial identification and classification, could be advantageously used for dereplication of recurrent bacterial isolates. In this study, we compared whole-cell MALDI-TOF MS-based analyses of 49 bacterial cultures to two well-established bacterial identification and classification methods based on nearly complete 16S rRNA gene sequence analyses: a phylotype-based approach, using a closest type strain assignment, and a sequence similarity-based approach involving a 98.65% sequence similarity threshold, which has been found to best delineate bacterial species. Culture classification using reference-based MALDI-TOF MS was comparable to that yielded by phylotype assignment up to the genus level. At the species level, agreement between 16S rRNA gene analysis and MALDI-TOF MS was found to be limited, potentially indicating that spectral reference databases need to be improved. We also evaluated the mass spectral similarity technique for species-level delineation which can be used independently of reference databases. We established optimal mass spectral similarity thresholds which group MALDI-TOF mass spectra of common environmental isolates analogically to phylotype- and sequence similarity-based approaches. When using a mass spectrum similarity approach, we recommend a mass range of 4-10 kDa for analysis, which is populated with stable mass signals and contains the majority of phylotype-determining peaks. We show that a cosine similarity (CS) threshold of 0.79 differentiate mass spectra analogously to 98.65% species-level delineation sequence similarity threshold, with corresponding precision and recall values of 0.70 and 0.73, respectively. When matched to species-level phylotype assignment, an optimal CS threshold of 0.92 was calculated, with associated precision and recall values of 0.83 and 0.64, respectively. Overall, our research indicates that a similarity-based MALDI-TOF MS approach can be routinely used for efficient dereplication of isolates for downstream analyses, with minimal loss of unique organisms. In addition, MALDI-TOF MS analysis has further improvement potential unlike 16S rRNA gene analysis, whose methodological limits have reached a plateau.

Murine Plasma N-Glycosylation Traits Associated with Sex and Strain.

Glycosylation is an abundant and important protein modification with large influence on the properties and interactions of glycoconjugates. Human plasma N-glycosylation has been the subject of frequent investigation, revealing strong associations with physiological and pathological conditions. Less well-characterized is the plasma N-glycosylation of the mouse, the most commonly used animal model for studying human diseases, particularly with regard to differences between strains and sexes. For this reason, we used MALDI-TOF(/TOF)-MS(/MS) assisted by linkage-specific derivatization of the sialic acids to comparatively analyze the plasma N-glycosylation of both male and female mice originating from BALB/c, CD57BL/6, CD-1, and Swiss Webster strains. The combined use of this analytical method and the recently developed data processing software named MassyTools allowed the relative quantification of the N-glycan species within plasma, the distinction between α2,3- and α2,6-linked N-glycolylneuraminic acids (due to respective lactonization and ethyl esterification), the detection of sialic acid O-acetylation, as well as the characterization of branching sialylation (Neu5Gcα2,3-Hex-[Neu5Gcα2,6-]HexNAc). When analyzing the glycosylation according to mouse sex, we found that female mice present a considerably higher degree of core fucosylation (2-4-fold depending on the strain), galactosylation, α2,6-linked sialylation, and larger high-mannose type glycan species compared with their male counterparts. Male mice, on the contrary, showed on average higher α2,3-linked sialylation, branching sialylation, and putative bisection. These differences together with sialic acid acetylation proved to be strain-specific as well. Interestingly, the outbred strains CD-1 and Swiss Webster displayed considerably larger interindividual variation than inbred strains BALB/c and CD57BL/6, suggesting a strong hereditable component of the observed plasma N-glycome.

Renopathological Microstructure Visualization from Formalin Fixed Kidney Tissue by Matrix-Assisted Laser/Desorption Ionization-Time-of-Flight Mass Spectrometry Imaging.

Understanding early stage renal malfunctions with regard to the glomerular filtration processes is essential for nephropathological prescreening strategies and intervention at an early stage. Mass spectrometry imaging (MSI) in combination with histopathology can provide an universal analytical approach. Proteomic and lipidomic aspects of glomerular biocompositions were applied for micro-structural differentiation in healthy rat kidney samples. Usability of commonly used tissue embedding media and the compatibility of histological staining and fixation methods were of interest. It was demonstrated that ultra-thin tissue samples (500 nm, 1 and 10 µm) can be used for lipid and peptide-based differentiation at the glomerular resolution level in formalin-fixed tissue samples in combination with preceding histological staining for correlating optical and molecular mass images.