Rationale: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. Methods: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2O. Results: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Conclusion: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors. Published 2024. This article is a U.S. Government work and is in the public domain in the USA.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder caused by pathogenic variants in the ATP-binding cassette subfamily D member 1 gene (ABCD1) that encodes the adrenoleukodystrophy protein (ALDP). Defects in ALDP result in elevated cerotic acid, and lead to C26:0-lysophosphatidylcholine (C26:0-LPC) accumulation, which is the primary biomarker used in newborn screening (NBS) for X-ALD. C26:0-LPC levels were measured in dried blood spot (DBS) NBS specimens using a flow injection analysis (FIA) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) performed in negative ion mode. The method was validated by assessing and confirming linearity, accuracy, and precision. We have also established C26:0-LPC cutoff values that identify newborns at risk for X-ALD. The mean concentration of C26:0-LPC in 5881 de-identified residual routine NBS specimens was 0.07 ± 0.02 µM (mean + 1 standard deviation (SD)). All tested true X-ALD positive and negative samples were correctly identified based on C26:0-LPC cutoff concentrations for borderline between 0.15 µM and 0.22 µM (mean + 4 SD) and presumptive screening positive at ≥0.23 µM (mean + 8 SD). The presented FIA method shortens analysis run-time to 1.7 min, while maintaining the previously established advantage of utilizing negative mode MS to eliminate isobaric interferences that could lead to screening false positives.

OBJECTIVES: The goal of this study was to include the quantitation of hexacosanoyl lysophosphatidylcholine, a biomarker for X-linked adrenoleukodystrophy and other peroxisomal disorders, in the routine extraction and analysis procedure used to quantitate amino acids, acylcarnitines, and succinylacetone during newborn screening. Criteria for the method included use of a single punch from a dried blood spot, one simple extraction of the punch, no high-performance liquid chromatography, and utilizing tandem mass spectrometry to quantitate the analytes. DESIGN AND METHODS: Dried blood spot punches were extracted with a methanolic solution of stable-isotope labeled internal standards, formic acid, and hydrazine, followed by flow injection analysis-electrospray ionization-tandem mass spectrometry. RESULTS: Quantitation of amino acids, acylcarnitines, and hexacosanoyl lysophosphatidylcholine using this combined method was similar to results obtained using two separate methods. CONCLUSIONS: A single dried blood spot punch extracted by a rapid (45min), simple procedure can be analyzed with high throughput (2min per sample) to quantitate amino acids, acylcarnitines, succinylacetone, and hexacosanoyl lysophosphatidylcholine.

BACKGROUND: 3-Hydroxypalmitoleoyl-carnitine (C16:1-OH) has recently been reported to be elevated in acylcarnitine profiles of patients with propionic acidemia (PA) or methylmalonic acidemia (MMA) during expanded newborn screening (NBS). High levels of C16:1-OH, combined with other hydroxylated long chain acylcarnitines are related to long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and trifunctional protein (TFP) deficiency. METHODS: The acylcarnitine profile of two LCHADD patients was evaluated using liquid chromatography-tandem mass spectrometric method. A specific retention time was determined for each hydroxylated long chain acylcarnitine. The same method was applied to some neonatal dried blood spots (DBS) from PA and MMA patients presenting abnormal C16:1-OH concentrations. RESULTS: The retention time of the peak corresponding to C16:1-OH in LCHADD patients differed from those in MMA and PA patients. Heptadecanoylcarnitine (C17) has been identified as the novel biomarker specific for PA and MMA patients through high resolution mass spectrometry (Orbitrap) experiments. We found that 21 out of 23 neonates (22 MMA, and 1PA) diagnosed through the Tuscany region NBS program exhibited significantly higher levels of C17 compared to controls. Twenty-three maternal deficiency (21 vitamin B12 deficiency, 1 homocystinuria and 1 gastrin deficiency) samples and 82 false positive for elevated propionylcarnitine (C3) were also analyzed. CONCLUSIONS: We have characterized a novel biomarker able to detect propionate disorders during expanded newborn screening (NBS). The use of this new biomarker may improve the analytical performances of NBS programs especially in laboratories where second tier tests are not performed.

OBJECTIVES: Newborn screening for X-linked adrenoleukodystrophy utilizes tandem mass spectrometry to analyze dried-blood spot specimens. Quality control materials (dried-blood spots enriched with hexacosanoyl lysophosphatidylcholine) were prepared and stored at different temperatures for up to 518days to evaluate the stability of this biomarker for X-linked adrenoleukodystrophy. DESIGN AND METHODS: Dried-blood spot storage included desiccant (45, 171, and 518days) or omitted desiccant (53days at >90% relative humidity). Specimens were stored for 171 and 518days at -20 degrees C, 4 degrees C, ambient temperature, and 37 degrees C. Each weekday for 45days, a bag of specimens stored at 4 degrees C was warmed to ambient temperature and one specimen was removed for storage at -80 degrees C. Specimens were analyzed by high-performance liquid-chromatography electrospray ionization tandem mass spectrometry and data was plotted as concentration (micromoles per liter) vs. time. Linear regression provided slope and y-intercept values for each storage condition. RESULTS: Small slope values (0.01 or less) and y-intercept values close to the enrichment indicated less than 11% loss of hexacosanoyl lysophosphatidylcholine under all storage conditions tested. CONCLUSIONS: Quality control materials for X-linked adrenoleukodystrophy are stable for at least 1year when stored with desiccant.

BACKGROUND: Orotic aciduria in the presence of hyperammonemia is a key indicator for a defect in the urea cycle, specifically ornithine transcarbamylase (OTC) deficiency. Current newborn screening (NBS) protocols can detect several defects of the urea cycle, but screening for OTC deficiency remains a challenge due to the lack of a suitable assay. The purpose of this study was to develop a high-throughput assay to measure orotic acid in dried blood spot (DBS) specimens as an indicator for urea cycle dysfunction, which can be readily incorporated into routine NBS. METHODS: Orotic acid was extracted from DBS punches and analyzed using flow-injection analysis tandem mass spectrometry (FIA-MS/MS) with negative-mode ionization, requiring <2min/sample run time. This method was then multiplexed into a conventional newborn screening assay for analysis of amino acids, acylcarnitines, and orotic acid. RESULTS: We describe 2 assays which can quantify orotic acid in DBS: a stand-alone method and a combined method for analysis of orotic acid, amino acids, and acylcarnitines. Both methods demonstrated orotic acid recovery of 75-85% at multiple levels of enrichment. Precision was also comparable to traditional FIA-MS/MS methods. Analysis of residual presumptively normal NBS specimens demonstrated a 5:1 signal to noise ratio and the average concentration of orotic acid was approximately 1.2mumol/l. The concentration of amino acids and acylcarnitines as measured by the combined method showed no significant differences when compared to the conventional newborn screening assay. In addition, retrospective analysis of confirmed patients and presumptively normal newborn screening specimens suggests potential for the methods to identify patients with OTC deficiency, as well as other urea cycle defects. CONCLUSION: The assays described here quantify orotic acid in DBS using a simple extraction and FIA-MS/MS analysis procedures that can be implemented into current NBS protocols.

Sickle cell diseases are inborn blood disorders caused by the presence of an abnormal form of hemoglobin, hemoglobin S (HbS). Mortality from sickle cell disease during the first 3 to 4 years of life can be virtually eliminated by newborn screening and appropriate follow-up and treatment [1]. Three sickle cell diseases (sickle cell anemia, sickle hemoglobin C disease and sickle β thalassemia) are included in the United States' recommended uniform newborn screening panel [2]. HbS is the newborn screening marker for all three disorders. | The Newborn Screening Quality Assurance Program of the Centers for Disease Control and Prevention (CDC) prepares and validates dried-blood spot (DBS) proficiency testing materials to assist laboratories with monitoring the performance of their newborn screening tests [3]. As part of routine evaluations of marker stabilities in DBS samples, we used measurements by high performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) methods to compare the stabilities of HbA and HbS in DBSs stored for predetermined intervals at elevated temperature (37°C) and low (<30%) or high (>50%) humidity. The objectives of these studies were to measure separately the contributions of heat and humidity to the degradation of HbS and HbA in DBS samples and to evaluate the level of concordance between results from the two analysis methods used.

BACKGROUND: Hemoglobinopathies are mutations resulting in abnormal globin chain structure; some have clinically significant outcomes such as anemia or reduced lifespan. Five beta-globin mutations are (c.20A>T, p.E6V), (c.19G>A, p. E6K), (c.79G>A, p.E26K), (c.364G>C, p.E121Q), and (c.364G>A, p.E121K), resulting in HbS (sickle-cell hemoglobin), HbC, HbE, HbD-Los Angeles, and HbO-Arab, respectively. One alpha-globin mutation is (c.[207C>G or 207C>A], p.N68K), resulting in HbG-Philadelphia. METHODS: HPLC-ESI-MS/MS analysis of dried-blood spot (DBS) punches from newborns extracted with a trypsin-containing solution provides greater than 90% coverage of alpha-, beta-, and gamma-globin amino acid sequences. Because the (c.20A>T, p.E6V), (c.19G>A, p. E6K), (c.79G>A, p.E26K), (c.364G>C, p.E121Q), (c.364G>A, p.E121K), and (c.[207C>G or 207C>A], p.N68K) mutations generate globin peptides with novel amino acid sequences, detecting one of these peptides in DBS extracts is indicative of the presence of a hemoglobinopathy in the newborn. RESULTS: The method described here can distinguish normal beta-globin peptides from the mutant HbS, HbC, HbE, HbD-Los Angeles and HbO-Arab peptides, as well as normal alpha-globin peptide from the mutant HbG-Philadelphia peptide, allowing the identification of unaffected heterozygotes such as HbAS, and of compound heterozygotes such as HbASG-Philadelphia. CONCLUSIONS: This HPLC-ESI-MS/MS analytical approach provides information that is not available from traditional hemoglobin analyses such as isoelectric focusing and HPLC-UV. It is also capable of determining the amino acid sequence of hemoglobin peptides, potentially allowing the detection of numerous hemoglobinopathies resulting from point mutations.

BACKGROUND: Succinylacetone (SUAC) is the primary metabolic marker for hepatorenal tyrosinemia. MATERIALS AND METHODS: We used results reported for dried-blood-spot proficiency testing (PT) specimens and hepatorenal tyrosinemia patients' newborn screening (NBS) samples to demonstrate analytic biases in SUAC recoveries and differences in presumptive clinical classifications. RESULTS: SUAC recoveries from non-kit and NeoBase kit tandem mass spectrometry methods were markedly different. Kit users that set high cutoff values submitted discordant clinical assessments of "within normal limits" for PT specimens enriched with 10-15mcmol SUAC/L in blood. SUAC levels in tyrosinemia patients' NBS samples analyzed by NeoBase kit were lower than those in samples analyzed by non-kit methods. CONCLUSIONS: From 2009 to 2011, analytic biases in SUAC recoveries were consistent. Discordant clinical assessments of PT specimens were associated with high cutoff values for NeoBase kit results. Method-related differences in SUAC concentrations of tyrosinemia patients' samples were consistent with those of PT specimens.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is the most common human peroxisomal disorder, and is caused by mutations in the peroxisomal transmembrane ALD protein (ALDP, ABCD1). The biochemical defect associated with X-ALD is an accumulation of very long-chain fatty acids (VLCFA, e.g. C24:0 and C26:0), which has been shown to result in the accumulation of C26:0-lysophosphatidylcholine (C26:0-LPC). METHODS: We describe the analysis of C26:0-LPC in dried-blood spots (DBS) using a rapid (30min) and simple extraction procedure, isocratic HPLC resolution of LPC, and structure-specific analysis via negative ion mode tandem mass spectrometry. RESULTS: In putative normal DBS specimens from newborns (N=223) C26:0-LPC was 0.09+/-0.03mcmol/l whole blood, while in peroxisomal biogenesis disorder (including X-ALD) patients (N=28) C26:0-LPC was 1.13+/-0.67mcmol/l whole blood. Both multiple reaction monitoring and a neutral loss scan (225.1Da) analysis of DBS were used to analyze LPC. CONCLUSIONS: Compared to a previous report of C26:0-LPC analysis in DBS, the method described here is simpler, faster, and more structure-specific for LPC with C26:0 acyl chains.

This study describes the use of a stable-isotope labeled precursor ([U-13C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry, and factors to consider in interpreting the data, which include the isotope's location (13C appears in three isotopomers and isotopologues: [M + 16 for the sphingoid base or N-acyl fatty acid, and [M + 32] for both), the isotopic enrichment of palmitoyl-CoA, and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-13C]-palmitic acid, ~60% of the total palmitoyl-CoA was 13C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, -monohexosylceramide and -sphingomyelin were 62 + 3, 13 + 2, and 60 + 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 + 11 pmol/h per mg protein). Including estimates for the very-long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ~1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has heretofore been possible, while acknowledging that there are inherent limitations to such approximations.

Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo(2)-lipid A (KLA), causes a large increase in cellular sphingolipids, from 1.5 to 2.6 x 10(9) molecules per cell in 24 h, based on the sum of subspecies analyzed by "lipidomic" mass spectrometry. Thus, this study asked the following question. What is the cause of this increase and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis based on [U-(13)C]palmitate labeling, inhibition by ISP1 (myriocin), and an apparent induction of many steps of the pathway (according to the distribution of metabolites and microarray analysis), with the exception of ceramide, which is also produced from pre-existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.

BACKGROUND: Methionine (Met) is a key metabolite used in the newborn screening of homocystinuria by tandem mass spectrometry (MS/MS). Recently, a loss of ion counts in both Met and its deuterium-labeled internal standard ((2)H(3)-Met) was observed by the CDC's Newborn Screening Quality Assurance Program laboratory. We report on the stability of labeled and unlabeled Met solutions and their storage in two types of 96 well microtiter plates to illustrate the potential loss of Met following storage of samples prior to MS/MS analysis. METHODS: Neat labeled and unlabeled Met standards were prepared and added (25, 50 and 100 microl) to two different types of microtiter plates, dried under nitrogen and stored for up to 168 h. All samples were reconstituted in mobile phase and analyzed as free acids for simplification of the study. RESULTS AND CONCLUSIONS: Met appears to interact significantly with polystyrene microtiter plates and to a much lesser extent with polypropylene microtiter plates. Furthermore, the loss is greatest for lower concentrations of methionine. While this loss of Met signal may be unimportant due to a presumption of equal loss of (2)H(3)-Met, a significant decline in ion signals will cause greater error in the calculation of concentration. These results suggest that polypropylene may be a better choice for Met analysis. Furthermore, storing prepared samples prior to analysis may impact the quality of the MS/MS analysis for Met and potentially other metabolites. Plates used by newborn screening laboratories should be evaluated periodically if the signal intensity for Met is reduced.