Immuno‐MALDI‐TOF MS: New perspectives for clinical applications of mass spectrometry

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.     

Rapid identification and characterization of Vibrio species using whole‐cell MALDI‐TOF mass spectrometry

Aims: Vibrio identification by means of traditional microbiological methods is time consuming because of the many biochemical tests that have to be performed to distinguish closely related species. This work aimed at evaluating the use of MALDI‐TOF mass spectrometry for the rapid identification of Vibrio (V.) spp. as an advantageous application to rapidly discriminate the most important Vibrio spp. and distinguish Vibrio spp. from closely related bacterial species like Photobacterium damselae and Grimontia hollisae and other aquatic bacteria like Aeromonas spp. Methods and Results: Starting from sub‐colony amounts of pure cultures grown on agar plates, a very simple sample preparation procedure was established and combined with a rapid and automated measurement protocol that allowed species identification within minutes. Closely related species like Vibrio alginolyticus and Vibrio parahaemolyticus or Vibrio cholerae and Vibrio mimicus could thus be differentiated by defining signatures of species‐identifying biomarker ions (SIBIs). As a reference method for species designation and for determination of relationships between strains with molecular markers, partial rpoB gene sequencing was applied. Conclusions: The MALDI‐TOF MS‐based method as well as the rpoB sequence‐based approach for Vibrio identification described in this study produced comparable classification results. The construction of phylogenetic trees from MALDI‐TOF MS and rpoB sequences revealed a very good congruence of both methods. Significance and Impact of the Study: Our results suggest that whole‐cell MALDI‐TOF MS‐based proteometric characterization represents a powerful tool for rapid and accurate classification and identification of Vibrio spp. and related species.     

Comparative analysis of storage conditions and homogenization methods for tick and flea species for identification by MALDI‐TOF MS

Ticks and fleas are vectors for numerous human and animal pathogens. Controlling them, which is important in combating such diseases, requires accurate identification, to distinguish between vector and non‐vector species. Recently, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was applied to the rapid identification of arthropods. The growth of this promising tool, however, requires guidelines to be established. To this end, standardization protocols were applied to species of Rhipicephalus sanguineus (Ixodida: Ixodidae) Latreille and Ctenocephalides felis felis (Siphonaptera: Pulicidae) Bouché, including the automation of sample homogenization using two homogenizer devices, and varied sample preservation modes for a period of 1–6 months. The MS spectra were then compared with those obtained from manual pestle grinding, the standard homogenization method. Both automated methods generated intense, reproducible MS spectra from fresh specimens. Frozen storage methods appeared to represent the best preservation mode, for up to 6 months, while storage in ethanol is also possible, with some caveats for tick specimens. Carnoy's buffer, however, was shown to be less compatible with MS analysis for the purpose of identifying ticks or fleas. These standard protocols for MALDI‐TOF MS arthropod identification should be complemented by additional MS spectrum quality controls, to generalize their use in monitoring arthropods of medical interest.     

Effect of trapping method on species identification of phlebotomine sandflies by MALDI‐TOF MS protein profiling

Sandflies (Diptera: Psychodidae) (Newstead, 1911) are blood‐feeding insects that transmit human pathogens including Leishmania (Trypanosomatida: Trypanosomatidae) parasites, causative agents of the leishmaniases. To elucidate Leishmania transmission cycles, conclusive identification of vector species is essential. Molecular approaches including matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) protein profiling have recently emerged to complement morphological identification. The aim of this study was to evaluate the effect of the trap type used to collect sandflies, specifically Centers for Disease Control (CDC) light or sticky traps, the two most commonly used in sandfly surveys, on subsequent MALDI‐TOF MS protein profiling. Specimens of five species (Phlebotomus ariasi, Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus sergenti, Sergentomyia minuta) collected in periurban and agricultural habitats in southeast Spain were subjected to protein profiling. Acquired protein spectra were queried against an in‐house reference database and their quality assessed to evaluate the trap type effect. The results indicate that trap choice can substantially affect the quality of protein spectra in collected sandflies. Whereas specimens retrieved from light traps produced intense and reproducible spectra that allowed reliable species determination, profiles of specimens from sticky traps were compromised and often did not enable correct identification. Sticky traps should therefore not be used in surveys that deploy MALDI‐TOF MS protein profiling for species identification.     

On‐target and nanoparticle‐facilitated selective enrichment of peptides and proteins for analysis by MALDI‐MS

Over the course of the last decade, a number of investigators have come to appreciate that the surface of a MALDI target, after suitable modification, can be used for selective enrichment of peptides and proteins. More recently, surface‐modified nanoparticles (NPs) that readily co‐crystallize in MALDI matrix, are not ionized by laser desorption/ionization, and do not interfere with MS have attracted interest as alternatives to surface‐modified targets for selective enrichment of peptides and proteins. Surface‐modified targets and NPs facilitate parallel processing of samples, and when used in conjunction with MALDI mass spectrometers with kHz lasers enable development of high‐throughput proteomics platforms. Targets and NPs for reversed phase and ion exchange retention, selective enrichment of glycopeptides, selective enrichment of phosphopeptides, and immunoaffinity MS are described in conjunction with details regarding their preparation and utility. Commercial availability of the reagents and substrates required to prepare surface‐modified targets and NPs is also discussed.     

Identification of cross‐reactive B‐cell epitopes between Bos d 9.0101(Bos Taurus) and Gly m 5.0101 (Glycine max) by epitope mapping MALDI‐TOF MS

Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross‐allergenicity described between soy and milk proteins. We have previously identified several cross‐reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1‐casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α‐casein‐specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross‐reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI‐TOF MS analysis. On a second approach, the peptide mixture was resolved by RP‐HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI‐TOF MS. This novel MS based approach led us to identify and characterize four peptides on α‐casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross‐reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross‐reactivity, to further develop new and more effective vaccines for food allergy.     

Rapid characterization of N‐linked glycans from secreted and gel‐purified monoclonal antibodies using MALDI‐ToF mass spectrometry

Recombinant monoclonal antibodies (MAbs) are increasingly being used for therapeutic use and correct glycosylation of these MAbs is essential for their correct function. Glycosylation profiles are host cell‐ and antibody class‐dependent and can change over culture time and environmental conditions. Therefore, rapid monitoring of glycan addition/status is of great importance for process validity. We describe two workflows of generally applicability for glycan profiling of purified and gel‐purified MAbs produced in NS0 and CHO cells, in which small‐scale antibody purification and buffer exchange is combined with PNGase F glycan cleavage and graphite HyperCarb desalting. MALDI‐ToF mass spectrometry is used for sensitive detection of glycan forms, with the ability to confirm glycan structures by selective ion fragmentation. Both workflows are rapid, technically simple and amenable to automation, and use in multi‐well formats. Biotechnol. Bioeng. 2010;107: 902–908. © 2010 Wiley Periodicals, Inc.     

Improving peptide relative quantification in MALDI‐TOF MS for biomarker assessment

Proteomic profiling by MALDI‐TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI‐TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI‐TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI‐TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).     

EZYprep LC‐coupled MALDI‐TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.     

Enhanced detection of in‐gel released N‐glycans by MALDI‐TOF‐MS

Many biologically relevant glycoproteins need to be separated on 1D‐ or 2D‐gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in‐gel digestions with MALDI‐TOF‐MS. In this technical report, we investigated how the detection of in‐gel released N‐glycans could be improved by MALDI‐TOF‐MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D‐arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl‐β‐glucopyranoside, a nonionic detergent, was studied during in‐gel peptide‐N⁴‐(acetyl‐ß‐glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl‐β‐glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N‐glycans. A LOD of under 7 pmol glycoprotein could be achieved.     

MALDI‐TOF MS identification of microbiota associated with pest insect Diabrotica speciosa

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Usefulness and accuracy of MALDI‐TOF mass spectrometry as a supplementary tool to identify mosquito vector species and to invest in development of international database

Arthropod‐borne diseases are important causes of morbidity and mortality. The identification of vector species relies mainly on morphological features and/or molecular biology tools. The first method requires specific technical skills and may result in misidentifications, and the second method is time‐consuming and expensive. The aim of the present study is to assess the usefulness and accuracy of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a supplementary tool with which to identify mosquito vector species and to invest in the creation of an international database. A total of 89 specimens belonging to 10 mosquito species were selected for the extraction of proteins from legs and for the establishment of a reference database. A blind test with 123 mosquitoes was performed to validate the MS method. Results showed that: (a) the spectra obtained in the study with a given species differed from the spectra of the same species collected in another country, which highlights the need for an international database; (b) MALDI‐TOF MS is an accurate method for the rapid identification of mosquito species that are referenced in a database; (c) MALDI‐TOF MS allows the separation of groups or complex species, and (d) laboratory specimens undergo a loss of proteins compared with those isolated in the field. In conclusion, MALDI‐TOF MS is a useful supplementary tool for mosquito identification and can help inform vector control.     

Labeling elastase digests with TMT: informational gain by identification of poorly detectable peptides with MALDI-TOF/TOF mass spectrometry

The applicability of the less specific protease elastase for the identification of membrane and cytosolic proteins has already been demonstrated. MALDI as ionization technique particularly favors the detection of basic and to a lesser extent of weakly acidic peptides, whereas neutral peptides often remain undetected. Moreover, peptides below 700 Da are routinely excluded. In the following study, the advantage of additional information gained from tandem mass tag zero labeled peptides and the resultant increase in sequence coverage was evaluated. Through derivatization with tandem mass tag reagents, peptide measurement within the standard mass range of the MALDI reflector mode is achievable due to the mass increase. Compared to the unlabeled sample, peptides exhibiting relatively low molecular masses, pI values or higher hydrophobicity could be identified.