MALDI-MS in first-line screening of newborns for sickle cell disease: results from a prospective study in comparison to HPLC.

OBJECTIVES: Newborn screening (NBS) for sickle cell disease (SCD) requires a robust, high-throughput method to detect hemoglobin S (HbS). Screening for SCD is performed by qualitative methods, such as isoelectric focusing (IEF), and both qualitative and quantitative methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and tandem mass spectrometry (MS/MS). All these methods detect HbS, as well as low-level or absent HbA, and also other variants of hemoglobin. HPLC is considered as a reference method for NBS, because of its high sensitivity and specificity in detecting HbS. NeoSickle®, a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, combined with automated sample processing, a laboratory information management system and NeoSickle® software for automatic data interpretation, has increased the throughput of SCD testing. The purpose of this study was to compare the performances of NeoSickle® and HPLC. METHODS: A prospective study was conducted including 9,571 samples from the NBS program to compare MALDI-MS using NeoSickle® with an HPLC method. Correlation between the two methods was studied. For the MALDI-MS method, sensitivity, specificity, NPV, and PPV were calculated. RESULTS: We found over 99.4 % correlation between the HPLC and MALDI-MS results. NeoSickle® showed 100 % of sensitivity and specificity in detecting SCD syndrome, leading to positive and negative predictive values of 100 %. CONCLUSIONS: NeoSickle® is adapted to NBS for SCD, and can be used in first-line high-throughput screening to detect HbS, and beta-thalassemia major warning. When HbS is detected, second-line use of another specific method as HPLC is necessary.

A Multicentre Pilot Study of a Two-Tier Newborn Sickle Cell Disease Screening Procedure with a First Tier Based on a Fully Automated MALDI-TOF MS Platform.

The reference methods used for sickle cell disease (SCD) screening usually include two analytical steps: a first tier for differentiating haemoglobin S (HbS) heterozygotes, HbS homozygotes and ß-thalassemia from other samples, and a confirmatory second tier. Here, we evaluated a first-tier approach based on a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform with automated sample processing, a laboratory information management system and NeoSickle® software for automatic data interpretation. A total of 6701 samples (with high proportions of phenotypes homozygous (FS) or heterozygous (FAS) for the inherited genes for sickle haemoglobin and samples from premature newborns) were screened. The NeoSickle® software correctly classified 98.8% of the samples. This specific blood sample collection was enriched in qualified difficult samples (premature newborns, FAS samples, late and very late samples, etc.). In this study, the sensitivity of FS sample detection was found to be 100% on the Lille MS facility and 99% on the Dijon MS facility, and the specificity of FS sample detection was found to be 100% on both MS facilities. The MALDI-MS platform appears to be a robust solution for first-tier use to detect the HbS variant: it is reproducible and sensitive, it has the power to analyze 600-1000 samples per day and it can reduce the unit cost of testing thanks to maximal automation, minimal intervention by the medical team and good overall practicability. The MALDI-MS approach meets today's criteria for the large-scale, cost-effective screening of newborns, children and adults.

The Reliable, Automatic Classification of Neonates in First-Tier MALDI-MS Screening for Sickle Cell Disease.

Previous research has shown that a MALDI-MS technique can be used to screen for sickle cell disease (SCD), and that a system combining automated sample preparation, MALDI-MS analysis and classification software is a relevant approach for first-line, high-throughput SCD screening. In order to achieve a high-throughput "plug and play" approach while detecting "non-standard" profiles that might prompt the misclassification of a sample, we have incorporated various sets of alerts into the decision support software. These included "biological alert" indicators of a newborn's clinical status (e. g., detecting samples with no or low HbA), and "technical alerts" indicators for the most common non-standard profiles, i.e., those which might otherwise lead to sample misclassification. We evaluated these alerts by applying them to two datasets (produced by different laboratories). Despite the random generation of abnormal spectra by one-off technical faults or due to the nature and quality of the samples, the use of alerts fully secured the process of automatic sample classification. Firstly, cases of ß-thalassemia were detected. Secondly, after a visual check on the tagged profiles and reanalysis of the corresponding biological samples, all the samples were correctly reclassified without prompting further alerts. All of the samples for which the results were not tagged were well classified (i.e., sensitivity and specificity = 1). The alerts were mainly designed for detecting false-negative classifications; all the FAS samples misclassified by the software as FA (a false negative) were marked with an alert. The implementation of alerts in the NeoScreening® Laboratory Information Management System's decision support software opens up perspectives for the safe, reliable, automated classification of samples, with a visual check solely on abnormal results or samples. It should now be possible to evaluate the combination of the NeoSickle® analytical solution and the NeoScreening® Laboratory Information Management System in a real-life, prospective study of first-line SCD screening.

Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference.

Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two-day Pan-European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus-based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.

S-Nitrosylation of cIAP1 Switches Cancer Cell Fate from TNFα/TNFR1-Mediated Cell Survival to Cell Death.

TNFα is a prominent proinflammatory cytokine and a critical mediator for the development of many types of cancer such as breast, colon, prostate, cervical, skin, liver, and chronic lymphocytic leukemia. Binding of TNFα to TNFR1 can lead to divergent signaling pathways promoting predominantly NF-κB activation but also cell death. We report here that the nitric oxide (NO) donor glyceryl trinitrate (GTN) converts TNFα, generated from immune cells or cancer cells stimulated by chemotherapy, into a prodeath mediator in colon and mammary cancer cells. GTN-mediated S-nitrosylation of cIAP1 on cysteines 571 and 574 inhibited its E3 ubiquitin ligase activity, which in turn reduced Lys63-linked ubiquitination of RIP1 and initiated assembly of a death complex. These findings provide insights into how NO can harness advantageous aspects of inflammation in cancer and provide new therapeutic strategies.Significance: Combination of an NO donor with chemotherapeutic drug-induced TNFα represents a potentially valuable anticancer strategy. Cancer Res; 78(8); 1948-57. ©2018 AACR.

Variance component analysis to assess protein quantification in biomarker discovery. Application to MALDI-TOF mass spectrometry.

Controlling the technological variability on an analytical chain is critical for biomarker discovery. The sources of technological variability should be modeled, which calls for specific experimental design, signal processing, and statistical analysis. Furthermore, with unbalanced data, the various components of variability cannot be estimated with the sequential or adjusted sums of squares of usual software programs. We propose a novel approach to variance component analysis with application to the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology and use this approach for protein quantification by a classical signal processing algorithm and two more recent ones (BHI-PRO 1 and 2). Given the high technological variability, the quantification failed to restitute the known quantities of five out of nine proteins present in a controlled solution. There was a linear relationship between protein quantities and peak intensities for four out of nine peaks with all algorithms. The biological component of the variance was higher with BHI-PRO than with the classical algorithm (80-95% with BHI-PRO 1, 79-95% with BHI-PRO 2 vs. 56-90%); thus, BHI-PRO were more efficient in protein quantification. The technological component of the variance was higher with the classical algorithm than with BHI-PRO (6-25% vs. 2.5-9.6% with BHI-PRO 1 and 3.5-11.9% with BHI-PRO 2). The chemical component was also higher with the classical algorithm (3.6-18.7% vs. < 3.5%). Thus, BHI-PRO were better in removing noise from signal when the expected peaks are detected. Overall, either BHI-PRO algorithm may reduce the technological variance from 25 to 10% and thus improve protein quantification and biomarker validation.

Bayesian inference for biomarker discovery in proteomics: an analytic solution.

This paper addresses the question of biomarker discovery in proteomics. Given clinical data regarding a list of proteins for a set of individuals, the tackled problem is to extract a short subset of proteins the concentrations of which are an indicator of the biological status (healthy or pathological). In this paper, it is formulated as a specific instance of variable selection. The originality is that the proteins are not investigated one after the other but the best partition between discriminant and non-discriminant proteins is directly sought. In this way, correlations between the proteins are intrinsically taken into account in the decision. The developed strategy is derived in a Bayesian setting, and the decision is optimal in the sense that it minimizes a global mean error. It is finally based on the posterior probabilities of the partitions. The main difficulty is to calculate these probabilities since they are based on the so-called evidence that require marginalization of all the unknown model parameters. Two models are presented that relate the status to the protein concentrations, depending whether the latter are biomarkers or not. The first model accounts for biological variabilities by assuming that the concentrations are Gaussian distributed with a mean and a covariance matrix that depend on the status only for the biomarkers. The second one is an extension that also takes into account the technical variabilities that may significantly impact the observed concentrations. The main contributions of the paper are: (1) a new Bayesian formulation of the biomarker selection problem, (2) the closed-form expression of the posterior probabilities in the noiseless case, and (3) a suitable approximated solution in the noisy case. The methods are numerically assessed and compared to the state-of-the-art methods (t test, LASSO, Battacharyya distance, FOHSIC) on synthetic and real data from proteins quantified in human serum by mass spectrometry in selected reaction monitoring mode.

Peroxisomal ATP-binding cassette transporters form mainly tetramers.

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane.

Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease.

Better identification of severe acute graft-versus-host disease (GvHD) may improve the outcome of this life-threatening complication of allogeneic hematopoietic stem cell transplantation. GvHD induces tissue damage and the release of damage-associated molecular pattern (DAMP) molecules. Here, we analyzed GvHD patients (n = 39) to show that serum heat shock protein glycoprotein 96 (Gp96) could be such a DAMP molecule. We demonstrate that serum Gp96 increases in gastrointestinal GvHD patients and its level correlates with disease severity. An increase in Gp96 serum level was also observed in a mouse model of acute GvHD. This model was used to identify complement C3 as a main partner of Gp96 in the serum. Our biolayer interferometry, yeast two-hybrid and in silico modeling data allowed us to determine that Gp96 binds to a complement C3 fragment encompassing amino acids 749-954, a functional complement C3 hot spot important for binding of different regulators. Accordingly, in vitro experiments with purified proteins demonstrate that Gp96 downregulates several complement C3 functions. Finally, experimental induction of GvHD in complement C3-deficient mice confirms the link between Gp96 and complement C3 in the serum and with the severity of the disease.

Associations between food consumption patterns and saliva composition: Specificities of eating difficulties children.

Identifying objective markers of diet would be beneficial to research fields such as nutritional epidemiology. As a preliminary study on the validity of using saliva for this purpose, and in order to explore the relationship between saliva and diet, we focused on clearly contrasted groups of children: children with eating difficulties (ED) receiving at least 50% of their energy intake through artificial nutrition vs healthy controls (C). Saliva of ED and C children was analyzed by various methods (targeted biochemical analyses, 2-D electrophoresis coupled to MS, 1H NMR) and their diet was characterized using food frequency questionnaires, considering 148 food items grouped into 13 categories. Complete datasets were obtained for 16 ED and 16 C subjects (median age 4.7y and 5.0y, respectively) and the statistical link between salivary and dietary characteristics was studied by Multiple Factor Analysis (MFA). Overall, ED children showed as expected lower consumption frequency scores and higher food selectivity. The two groups of children differed in "diet/saliva" associations. Some distinctive salivary variables were common to both groups of children. For example, carbonic anhydrase 6 and the consumption frequency of biscuits & sweets and drinks were positively associated with the MFA axis 1 in C children, but oppositely associated in ED children. Specifically for ED children, abundant salivary proteins (cystatins, amylase, amylase fragments) and some metabolites (amino acids, galactose, lactate) correlated with axis 1, together with the consumption frequency of sauces & seasonings, bread & cereal products, ready-to-eat meals, fish, biscuits & sweets, drinks and potatoes. Specifically for C children, several proteins (serum albumin, haptoglobin, Igκ, apolipoprotein A-1, α-1 antitrypsin) correlated with axis 1, together with the consumption frequency of biscuits & sweets, milk & dairy products, drinks, fruit, meat and vegetables. This study demonstrates that the qualitative aspect of diet is linked to saliva composition, and that the associations between dietary consumption and salivary composition differ between groups of subjects with contrasted diets.

Specific enrichment of 2-arachidonoyl-lysophosphatidylcholine in carotid atheroma plaque from type 2 diabetic patients.

BACKGROUND AND AIMS: Diabetic patients are at high risk of stroke and coronary artery disease. Recent data suggest that arachidonic acid metabolism is altered in diabetic conditions and that these alterations contribute to accelerated atherosclerosis. Little is known about how these alterations affect the metabolism and the proportions of different lipid species within the atherosclerotic plaque. The aim of our study was to perform a targeted lipidomic analysis of human atherosclerotic lesions, with a specific focus on PUFA-containing lipid species, to reveal differences in the fatty-acid composition of plaque in diabetic patients compared with non-diabetic controls. METHODS: Carotid atheroma plaque samples were obtained from 31 diabetic and 48 non-diabetic patients undergoing carotid endarterectomy. Targeted lipidomic analysis was then performed to determine the fatty acid composition of major glycerophospholipids and cholesteryl ester species by liquid chromatography-tandem mass spectrometry. RESULTS: Atheroma plaques from diabetic patients were significantly enriched with 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) (2.3 ± 0.8% Vs. 1.8 ± 0.6% p = 0.0002). Multivariable logistic regression showed that an increased 2-AA-LPC level was independently associated with diabetes. Finally, a positive relationship was found between 2-AA-LPC and HbA1c levels. Interestingly, endothelial lipase and calcium independent PLA2 gamma which could account for the production of 2-AA-LPC were detected in carotid plaques by immunohistochemistry. CONCLUSIONS: 2-AA-LPC stands at the crossroads of major metabolic pathways that lead to the synthesis of bioactive molecules such as AA-derived eicosanoids, 2-AA-lysophosphatidic acid and 2-AA-glycerol. 2-AA-LPC therefore appears to be a promising molecule to investigate in the context of diabetes.

Multi-omics profiling reveals that eating difficulties developed consecutively to artificial nutrition in the neonatal period are associated to specific saliva composition.

Prolonged enteral or parenteral nutrition in neonatal periods sometimes results in eating difficulties persisting for years, with reduced food intake through the oral route and thereby reduced stimulation of the oral cavity. Aiming at describing the consequences on oral physiology, saliva of 21 children with eating difficulties (ED) was compared to that of 23 healthy controls, using various omics and targeted methods. Overall, despite heterogeneity within the groups (age, medication etc.), the three spectral methods (MALDI-TOF, SELDI-TOF, (1)H NMR) allowed discriminating ED and controls, confirming that oral stimulation by food intake plays a role in shaping the composition of saliva. Saliva of ED patients exhibited a lower antioxidant status and lower levels of the salivary protease inhibitors cystatins. Other discriminant features (IgA1, dimethylamine) may relate to modified oral and/or intestinal microbial ecology. Finally, salivary profiles of ED patients were partly comparable to those of subjects with exacerbated gustatory sensitivities, in particular with reduced abundance of cystatin SN and higher abundance of zinc-alpha-2-glycoprotein. Whether this translates taste hypersensitivity and contributes to the eating difficulties deserves further attention.

Toward the Identification of Two Glycoproteins Involved in the Stomatal Deregulation of Downy Mildew-Infected Grapevine Leaves.

Stomata remain abnormally opened and unresponsive to abscisic acid in grapevine leaves infected by downy mildew. This deregulation occurs from 3 days postinoculation and increases concomitantly with leaf colonization by the pathogen. Using epidermal peels, we demonstrated that the active compound involved in this deregulation is located in the apoplast. Biochemical assays showed that the active compound present in the apoplastic fluids isolated from Plasmopara viticola-infected grapevine leaves (IAF) is a CysCys bridge-independent, thermostable and glycosylated protein. Fractionation guided assays based on chromatography coupled to stomatal response and proteomic analysis allowed the identification of both plant and pathogen proteins in the active fraction obtained from IAF. Further in silico analysis and discriminant filtrations based on the comparison between predictions and experimental indications lead to the identification of two Vitis vinifera proteins as candidates for the observed stomatal deregulation.

Glycated albumin with loss of fatty acid binding capacity contributes to enhanced arachidonate oxygenation and platelet hyperactivity: relevance in patients with type 2 diabetes.

High plasma concentrations of nonesterified fatty acids (NEFAs), transported bound to serum albumin, are associated with type 2 diabetes (T2D). The effects of albumin on platelet function were investigated in vitro. Modifications of albumin, such as those due to glycoxidation, were found in patients with T2D, and the consequences of these modifications on biological mechanisms related to NEFA handling were investigated. Mass spectrometry profiles of albumin from patients with T2D differed from those from healthy control subjects. Diabetic albumin showed impaired NEFA binding capacity, and both structural and functional alterations could be reproduced in vitro by incubating native albumin with glucose and methylglyoxal. Platelets incubated with albumin isolated from patients with T2D aggregated approximately twice as much as platelets incubated with albumin isolated from healthy control subjects. Accordingly, platelets incubated with modified albumin produced significantly higher amounts of arachidonate metabolites than did platelets incubated with control albumin. We concluded that higher amounts of free arachidonate are made available for the generation of active metabolites in platelets when the NEFA binding capacity of albumin is blunted by glycoxidation. This newly described mechanism, in addition to hypoalbuminemia, may contribute to platelet hyperactivity and increased thrombosis, known to occur in patients with T2D.

Comparison of classification methods that combine clinical data and high-dimensional mass spectrometry data.

BACKGROUND: The identification of new diagnostic or prognostic biomarkers is one of the main aims of clinical cancer research. Technologies like mass spectrometry are commonly being used in proteomic research. Mass spectrometry signals show the proteomic profiles of the individuals under study at a given time. These profiles correspond to the recording of a large number of proteins, much larger than the number of individuals. These variables come in addition to or to complete classical clinical variables. The objective of this study is to evaluate and compare the predictive ability of new and existing models combining mass spectrometry data and classical clinical variables. This study was conducted in the context of binary prediction. RESULTS: To achieve this goal, simulated data as well as a real dataset dedicated to the selection of proteomic markers of steatosis were used to evaluate the methods. The proposed methods meet the challenge of high-dimensional data and the selection of predictive markers by using penalization methods (Ridge, Lasso) and dimension reduction techniques (PLS), as well as a combination of both strategies through sparse PLS in the context of a binary class prediction. The methods were compared in terms of mean classification rate and their ability to select the true predictive values. These comparisons were done on clinical-only models, mass-spectrometry-only models and combined models. CONCLUSIONS: It was shown that models which combine both types of data can be more efficient than models that use only clinical or mass spectrometry data when the sample size of the dataset is large enough.

Nutri-metabolomics applied to taste perception phenotype: human subjects with high and low sensitivity to taste of fat differ in salivary response to oleic acid.

Saliva has different functions in the mouth and is involved, for example, in taste perception. Saliva composition can also be modified rapidly by taste stimulation. It remains unclear, however, whether the perceived intensity of a tastant may modulate this response. Based on increasing evidence that fat can be perceived by the taste system and that fat taste perception may be associated with fat intake, the aim of this work was to study if stimulation by a fatty acid (oleic acid) modifies saliva composition differently in subjects highly (sensitive+) or weakly (sensitive-) sensitive to that taste. For that purpose, saliva of two groups of subjects was collected after stimulation by either a control emulsion or an emulsion containing 5.61 mM oleic acid. Saliva was analyzed by 2D electrophoresis and (1)H NMR spectroscopy. The results show that sensitive+ and sensitive- subjects differ in their salivary response in terms of proteome and metabolome composition. Oppositely to sensitive- subjects, sensitive+ subjects responded to oleic acid by increased abundance of polymeric immunoglobulin receptor, rab GDP dissociation inhibitor beta, and organic acids, and decreased abundance of metabolites characteristic of mucins. The results highlight that modification of saliva composition by taste stimulation may be modulated by taste perception.

Symplekin, a polyadenylation factor, prevents MOZ and MLL activity on HOXA9 in hematopoietic cells.

MOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its inhibition leads to a decrease of the HOXA9 protein level but not of Hoxa9 mRNA and to an over-recruitment of MOZ and MLL onto the HOXA9 promoter. Altogether, our results highlight the role of Symplekin in transcription repression involving a regulatory network between MOZ, MLL and Symplekin.

Profiling serologic biomarkers in cirrhotic patients via high-throughput Fourier transform infrared spectroscopy: toward a new diagnostic tool of hepatocellular carcinoma.

Identification of novel serum biomarkers of hepatocellular carcinoma (HCC) is needed for early-stage disease detection and to improve patients' survival. The aim of this study was to evaluate the potential of serum Fourier transform infrared (FTIR) spectroscopy for differentiating sera from cirrhotic patients with and without HCC. Serum samples were collected from 2 sets of patients: cirrhotic patients with HCC (n = 39) and without HCC (n = 40). The FTIR spectra (10 per sample) were acquired in the transmission mode, and data homogeneity was tested by cluster analysis to exclude outliers. After data preprocessing by extended multiplicative signal correction and principal component analysis, the Support Vector Machine (SVM) method was applied using a leave-one-out cross-validation algorithm to classify the spectra into 2 classes of cirrhotic patients with and without HCC. When SVM was applied to all spectra (n = 790), the sensitivity and the specificity for the diagnosis of HCC were, respectively, 82.02% and 82.5%. When applied to the subset of spectra excluding the outliers (n = 739), SVM classification led to a sensitivity and specificity of 87.18% and 85%, respectively. Using median spectra for each patient instead of all replicates, the sensitivity and specificity were 84.62% and 82.50%, respectively. The overall accuracy rate was 82%-86%. In conclusion, this study suggests that FTIR spectroscopy combined with advanced methods of pattern analysis shows potential for differentiating sera from cirrhotic patients with and without HCC.

Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals.

Conventional identification (CI) of yeasts is based on morphological, biochemical and/or immunological methods. Matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF or MT-MS) mass spectrometry has been proposed as a new method for the identification of microorganisms. This prospective study compared the performance of MT-MS and CI for the identification of yeasts isolated from clinical samples. Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA was used as the reference method in the analysis of a total of 1207 yeast isolates. Concordance between MT-MS and CI was observed for 1105 isolates (91.5%), while 74 isolates (6.1%) were misidentified. Molecular identification revealed that 73 of these 74 isolates were identified correctly by MT-MS and CI correctly identified the last one. Concordance between the two techniques was excellent for the medically-important species (98-100%), including the identification of closely-related species (Candida albicans/C. dubliniensis; C. inconspicua/C. norvegensis; C. parapsilosis/C. metapsilosis/C. orthopsilosis). Only 2.3% of isolates belonging to C. famata, C. lambica and C. magnoliae or to Geotrichum spp. and Trichosporon spp. were not identified by MT-MS. This investigation highlights the potential of MT-MS-based yeast identification as a reliable, time and cost-efficient alternative to CI.