Evaluation of triage tool for low-grade cartilage tumors: Four-quadrant approach.

BACKGROUND AND OBJECTIVES: The "four-quadrant approach" (FQA) for triage of benign enchondromas (E) and low-grade malignant chondrosarcomas (LGC) divides patients into treatment categories based on the presence or absence of pain and observation of aggressive or benign radiographic features. This article evaluates the usefulness of the FQA in predicting E versus LGC and operative versus nonoperative outcome. METHODS: Patients had working diagnosis of E or LGC, 1-year minimum follow-up, imaging, clinical data, outcomes, and no radiographic evidence of high-grade chondrosarcoma. Statistical analysis determined whether quadrant distribution correlated to E versus LGC and operative versus nonoperative intervention. RESULTS: Of 56 lesions (49 patients), 9 were LGC and 47 E. Twenty-five lesions (all 9 LGC, 16 E) were treated operatively and 31 (all E) nonoperatively. There were statistically significant correlations between quadrant distribution and both tumor type (p = 1.9 × 10-6 ) and operative intervention (p = 6.28 × 10-6 ). CONCLUSIONS: The FQA is a promising diagnostic tool to distinguish between E and LGC hyaline cartilage tumors, along with determining operative versus nonoperative intervention. Prospective evaluation is warranted.

Glycoprotein analysis using lectin microcolumns and capillary electrophoresis: Characterization of alpha1-acid glycoprotein by combined separation methods.

Separations based on combinations of 2.1 mm I.D. high-performance affinity microcolumns and capillary electrophoresis were developed and used to characterize the glycoforms of an intact glycoprotein. Human alpha1-acid glycoprotein (AGP) was used as a model analyte due to its heterogeneous glycosylation resulting from variations in its degree of branching, fucosylation, and number of sialic acids. Three separation formats were examined based on microcolumns that contained the lectins concanavalin A (Con A) or Aleuria aurantia lectin (AAL). These microcolumns were used with one another or in combination with capillary electrophoresis. N-Glycan analysis of the non-retained and retained AGP fractions was carried out by using PNGase F digestion and nanoflow electrospray ionization mass spectrometry. Con A microcolumns were found to selectively enrich AGP that contained bi-antennary N-glycans, while AAL microcolumns retained AGP with fucose-containing N-glycans. Results from these separation methods indicated that fucosylation of the N-linked glycans was more abundant when a high degree of branching was present in AGP. Sialic acid residues were more abundant when higher degrees of branching and more fucose residues were present in AGP. The separation and analysis methods that were developed could be used with relatively small amounts of AGP and can be adapted for use with other intact glycoproteins.

Ion mobility-resolved collision-induced dissociation and electron transfer dissociation of N-glycopeptides: gathering orthogonal connectivity information from a single mass-selected precursor ion population.

Glycopeptide-level mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses are commonly performed to establish site-specific protein glycosylation profiles that are of central importance to gaining structure-function insights on glycoproteins. Confoundingly, the complete characterization of glycopeptide connectivity usually requires the acquisition of multiple MS/MS fragmentation spectra. Complementary ion fragmentation techniques such as collision-induced dissociation (CID) and electron transfer dissociation (ETD) are often applied in concert to address this need. While structurally informative, the requirement for acquisition of two MS/MS spectra per analyte places considerable limitations upon the breadth and depth of large-scale glycoproteomic inquiry. Here, a previously developed method of multiplexing CID and ETD is applied to the study of glycopeptides for the first time. Integration of the two dissociation methods was accomplished through addition of an ion mobility (IM) dimension that disperses the two stages of MS/MS in time. This allows the two MS/MS spectra to be acquired within a few milliseconds of one another, and to be deconvoluted in post-processing. Furthermore, the method allows both fragmentation readouts to be obtained from the same precursor ion packet, thus reducing the inefficiencies imposed by separate CID and ETD acquisitions and the relatively poor precursor ion to fragment ion conversion typical of ETD. N-Linked glycopeptide ions ranging in molecular weight from 1.8 to 6.5 kDa were generated from four model glycoproteins that collectively encompassed paucimannosidic, high mannose, and complex types of N-glycosylation. In each case, IM-resolved CID and ETD events provided complete coverage of the glycan topology and peptide sequence coverages ranging from 48.4% (over 32 amino acid residues) to 85.7% (over eight amino acid residues). The potential of this method for large-scale glycoproteomic analysis is discussed.

A case for protein-level and site-level specificity in glycoproteomic studies of disease.

Abnormal glycosylation of proteins is known to be either resultant or causative of a variety of diseases. This makes glycoproteins appealing targets as potential biomarkers and focal points of molecular studies on the development and progression of human ailment. To date, a majority of efforts in disease glycoproteomics have tended to center on either determining the concentration of a given glycoprotein, or on profiling the total population of glycans released from a mixture of glycoproteins. While these approaches have demonstrated some diagnostic potential, they are inherently insensitive to the fine molecular detail which distinguishes unique and possibly disease relevant glycoforms of specific proteins. As a consequence, such analyses can be of limited sensitivity, specificity, and accuracy because they do not comprehensively consider the glycosylation status of any particular glycoprotein, or of any particular glycosylation site. Therefore, significant opportunities exist to improve glycoproteomic inquiry into disease by engaging in these studies at the level of individual glycoproteins and their exact loci of glycosylation. In this concise review, the rationale for glycoprotein and glycosylation site specificity is developed in the context of human disease glycoproteomics with an emphasis on N-glycosylation. Recent examples highlighting disease-related perturbations in glycosylation will be presented, including those involving alterations in the overall glycosylation of a specific protein, alterations in the occupancy of a given glycosylation site, and alterations in the compositional heterogeneity of glycans occurring at a given glycosylation site. Each will be discussed with particular emphasis on how protein-specific and site-specific approaches can contribute to improved discrimination between glycoproteomes and glycoproteins associated with healthy and unhealthy states.

Engaging challenges in glycoproteomics: recent advances in MS-based glycopeptide analysis.

The proteomic analysis of glycosylation is uniquely challenging. The numerous and varied biological roles of protein-linked glycans have fueled a tremendous demand for technologies that enable rapid, in-depth structural examination of glycosylated proteins in complex biological systems. In turn, this demand has driven many innovations in wide ranging fields of bioanalytical science. This review will summarize key developments in glycoprotein separation and enrichment, glycoprotein proteolysis strategies, glycopeptide separation and enrichment, the role of mass measurement accuracy in glycopeptide detection, glycopeptide ion dissociation methods for MS/MS, and informatic tools for glycoproteomic analysis. In aggregate, this selection of topics serves to encapsulate the present status of MS-based analytical technologies for engaging the challenges of glycoproteomic analysis.

Intermediate MCAD Deficiency Associated with a Novel Mutation of the ACADM Gene: c.1052C>T.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessive disorder that leads to a defect in fatty acid oxidation. ACADM is the only candidate gene causing MCAD deficiency. A single nucleotide change, c.985A>G, occurring at exon 11 of the ACADM gene, is the most prevalent mutation. In this study, we report a Caucasian family with multiple MCADD individuals. DNA sequence analysis of the ACADM gene performed in this family revealed that two family members showing mild MCADD symptoms share the same novel change in exon 11, c.1052C>T, resulting in a threonine-to-isoleucine change. The replacement is a nonconservative amino acid change that occurs in the C-terminal all-alpha domain of the MCAD protein. Here we report the finding of a novel missense mutation, c.1052C>T (p.Thr326Ile), in the ACADM gene. To our knowledge, c.1052C>T has not been previously reported in the literature or in any of the current databases we utilize. We hypothesize that this particular mutation in combination with p.Lys304Glu results in an intermediate clinical phenotype of MCADD.

Positive cell-free fetal DNA testing for trisomy 13 reveals confined placental mosaicism.

PURPOSE: We report on a case in which cell-free fetal DNA was positive for trisomy 13 most likely due to confined placental mosaicism. Cell-free fetal DNA testing analyzes DNA derived from placental trophoblast cells and can lead to incorrect results that are not representative of the fetus. METHODS: We sought to confirm commercial cell-free fetal DNA testing results by chorionic villus sampling and amniocentesis. These results were followed up by postnatal chromosome analysis of cord blood and placental tissue. RESULTS: First-trimester cell-free fetal DNA test results were positive for trisomy 13. Cytogenetic analysis of chorionic villus sampling yielded a mosaic karyotype of 47,XY,+13[10]/46,XY[12]. G-banded analysis of amniotic fluid was normal, 46,XY. Postnatal cytogenetic analysis of cord blood was normal. Karyotyping of tissues from four quadrants of the placenta demonstrated mosaicism for trisomy 13 in two of the quadrants and a normal karyotype in the other two. CONCLUSION: Our case illustrates several important aspects of this new testing methodology: that cell-free fetal DNA may not be representative of the fetal karyotype; that follow-up with diagnostic testing of chorionic villus sampling and/or amniotic fluid for abnormal test results should be performed; and that pretest counseling regarding the full benefits, limitations, and possible testing outcomes of cell-free fetal DNA screening is important.

Suspected brown recluse envenomation: a case report and review of different treatment modalities.

BACKGROUND: The Loxosceles reclusa, commonly known as the brown recluse spider, is responsible for virtually all cases of spider bites leading to a significant necrosis. CASE REPORT: We report the case of a 72-year-old man who presented to the Emergency Department complaining of back pain, weakness, and diarrhea. The patient stated that he sustained a bug bite 1 week before presenting to the hospital. His wound was necrotizing in nature and after an exhaustive work-up, the most likely etiology was found to be envenomation by a brown recluse spider, Loxosceles reclusa. CONCLUSION: This is an endemic cause of a necrotizing wound bite in areas of the Midwestern and Southern United States, but it is rarely reported in the Northeast.