Significant sensitivity improvements by matrix optimization: a MALDI-TOF mass spectrometric study of lipids from hen egg yolk

Due to its sensitivity, the tolerance of impurities and the simplicity of performance, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is increasingly used to analyze lipids from biological sources. Although its detailed role is not understood so far, the applied matrix has a pronounced effect on the achievable spectrum quality and particularly how sensitive the individual lipid classes are detectable. Different matrix compounds were recently established in the lipid field including 2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9-AA), para-nitroaniline (PNA), 2-mercaptobenzothiazole (MBT), and 2-(2-aminoethylamino)-5-nitropyridine (AAN). It is the aim of this paper to compare the properties of these matrices with the newly synthesized matrix, α-cyano-2,4-difluorocinnamic acid (Di-FCCA). An organic extract from hen egg yolk was used as a simple and easily available test system.It will be shown that Di-FCCA is the matrix of choice to detect lipids in the positive-ion mode due to an achievable sensitivity gain of more than one order of magnitude compared to alternative matrices. In contrast, Di-FCCA is not suitable for negative-ion detection of phospholipids. Here, 9-AA is unequivocally the matrix of choice.     

MALDI-TOF MS of phosphatidylethanolamines: Different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described.In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

The suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose?

Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound. Dedicated to Prof. Dr. Klaus Arnold on the occasion of his 65th birthday.     

Lipid analysis of human HDL and LDL by MALDI-TOF mass spectrometry and (31)P-NMR

The analysis of HDL and LDL is important for the further understanding of atherosclerosis because changes of the protein and lipid moieties occur under pathological conditions. Because destruction of lipids leads to the formation of well-defined products such as lysophospholipids or chlorohydrins, methods that allow their fast and reliable determination would be useful. In this study, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for the analysis of the lipid composition of human lipoproteins. These data were compared with high resolution (31)P-NMR spectroscopy. Differences between LDL and HDL in sphingomyelin and phosphatidylcholine content could be monitored by NMR and mass spectrometry, and differences with respect to the extraction efficiency were found by MALDI-TOF MS. Additionally, treatment of LDL with hypochlorite and phospholipase A(2) resulted in marked changes (formation of chlorohydrines and lysolipids). Lysophosphatidylcholines were detectable by both methods, whereas MALDI-TOF MS failed to detect chlorohydrines of phospholipids.We conclude that MALDI-TOF MS provides rapidly a reliable lipid profile of lipoproteins. However, a previous lipid separation must be performed to detect lipid oxidation products. NMR can be directly applied, but suffers from lower sensitivity, and provides only limited information on fatty acid composition.     

Identification of lipids in the cuticle of the parasitic nematode Anisakis simplex and the somatic tissues of the Atlantic cod Gadus morhua

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex, hexadecanoic acid in the dichloromethane extract of A. simplex, and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode. The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy:₁, CoCoPg and Bu0:0B:₆. The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex. Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite’s inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids - ceramides and sphingoid bases. The signals due to N-octanoyl-d-erythro-octasphinganine (m/z 288.3) and N-tetranoyl-d-erythro-tetradecasphinganine (m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.     

MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described. In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

利用基因组学和MALDI-TOF MS技术鉴定放线菌纲细菌的核糖体蛋白质标志物

【目的】基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)法基于微生物的特征蛋白指纹图谱鉴定菌种,本研究利用基因组学和MALDI-TOFMS技术鉴定放线菌纲细菌的核糖体蛋白质标志物。【方法】从MALDI-TOF MS图谱数据库选取放线菌纲代表菌种,在基因组数据库检索目标菌种,获取目标菌株或其参比菌株的核糖体蛋白质序列,计算获得分子质量理论值,用于注释目标菌株MALDI-TOFMS指纹图谱中的核糖体蛋白质信号。【结果】从8目,24科,53属,114种,142株放线菌的MALDI-TOFMS图谱中总共注释出31种核糖体蛋白质。各菌株的指纹图谱中核糖体蛋白质信号数量差异显著。各种核糖体蛋白质信号的注释次数差异显著。总共15种核糖体蛋白质在超过半数图谱中得到注释,注释次数最高的是核糖体大亚基蛋白质L36。【结论】本研究找到了放线菌纲细菌MALDI-TOF MS图谱中常见的15种核糖体蛋白质信号,可为通过识别核糖体蛋白质的质谱特征峰鉴定放线菌的方法建立提供依据。     

Rapid and robust MALDI-TOF MS techniques for microbial identification: a brief overview of their diverse applications

in mass spectrometry have enabled the investigation of various biological systems by directly analyzing diverse sets of biomolecules (i.e., proteins, lipids, and carbohydrates), thus making a significant impact on the life sciences field. Over the past decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely utilized as a rapid and reliable method for the identification of microorganisms. MALDI-TOF MS has come into widespread use despite its relatively low resolving power (full width at half maximum, FWHM: < 5,000) and its incompatibility with tandem MS analysis, features with which other high-resolution mass spectrometers are equipped. Microbial identification is achieved by searching databases containing mass spectra of peptides and proteins extracted from microorganisms of interest, using scoring algorithms to match analyzed spectra with reference spectra. In this paper, we give a brief overview of the diverse applications of rapid and robust MALDI-TOF MS-based techniques for microbial identification in a variety of fields, such as clinical diagnosis and environmental and food monitoring. We also describe the fundamental principles of MALDI-TOF MS. The general specifications of the two major MS-based microbial identification systems available in the global market (BioTyper® and VITEK® MS Plus) and the distribution of these instruments in Republic of Korea are also discussed. The current review provides an understanding of this emerging microbial identification and classification technology and will help bacteriologists and cell biologists take advantage of this powerful technique.     

An improved method for the analysis of Cryptosporidium parvum oocysts by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Cryptosporidium parvum oocysts were analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Sample preparation proved to be a crucial step in the acquisition of acceptable mass spectra. Oocysts of C. parvum and the matrix were mixed and held for at least 45 min to produce reproducible, representative mass spectra. Sporozoites were also excysted from oocysts, purified, and analyzed using MALDI-TOF MS. The mass spectra of the intact oocysts contained many of the same peaks found in the mass spectra of the sporozoites, suggesting that during analysis, the internal constituents, not just the oocyst wall, are ablated by the laser.     

Triacylglycerol profiling of marine microalgae by mass spectrometry

We present a method for the determination of triacylglycerol (TAG) profiles of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species (Phaeodactylum tricornutum, Nannochloropsis salina, Nannochloropsis oculata, and Tetraselmis suecica) using MALDI time-of-flight (TOF) mass spectrometry (MS), ESI linear ion trap-orbitrap (LTQ Orbitrap) MS, and 1H NMR spectroscopy. Direct MS analysis is particularly effective to compare the polyunsaturated fatty acid (PUFA) composition for triacylglycerols because oxidation can often degrade samples upon derivatization. Using these methods, we observed that T. suecica contains significant PUFA levels with respect to other microalgae. This method is applicable for high-throughput MS screening of microalgae TAG profiles and may aid in the commercial development of biofuels.     

The C-Terminal Fragment of Prostate-Specific Antigen,a 2331 Da Peptide,as a New Urinary Pathognomonic Biomarker Candidate for Diagnosing Prostate Cancer

Background and Objectives

Prostate cancer (PCa) is one of the most common cancers and leading cause of cancer-related deaths in men. Mass screening has been carried out since the 1990s using prostate-specific antigen (PSA) levels in the serum as a PCa biomarker. However, although PSA is an excellent organ-specific marker, it is not a cancer-specific marker. Therefore, the aim of this study was to discover new biomarkers for the diagnosis of PCa.

Materials and Methods

We focused on urine samples voided following prostate massage (digital rectal examination [DRE]) and conducted a peptidomic analysis of these samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MSⁿ). Urinary biomaterials were concentrated and desalted using CM-Sepharose prior to the following analyses being performed by MALDI-TOF/MSⁿ: 1) differential analyses of mass spectra; 2) determination of amino acid sequences; and 3) quantitative analyses using a stable isotope-labeled internal standard.

Results

Multivariate analysis of the MALDI-TOF/MS mass spectra of urinary extracts revealed a 2331 Da peptide in urine samples following DRE. This peptide was identified as a C-terminal PSA fragment composed of 19 amino acid residues. Moreover, quantitative analysis of the relationship between isotope-labeled synthetic and intact peptides using MALDI-TOF/MS revealed that this peptide may be a new pathognomonic biomarker candidate that can differentiate PCa patients from non-cancer subjects.

Conclusion

The results of the present study indicate that the 2331 Da peptide fragment of PSA may become a new pathognomonic biomarker for the diagnosis of PCa. A further large-scale investigation is currently underway to assess the possibility of using this peptide in the early detection of PCa.     

MALDI-TOF mass spectrometry in the clinical mycology laboratory: identification of fungi and beyond

MALDI-TOF mass spectrometry (MS) is becoming essential in most clinical microbiology laboratories throughout the world. Its successful use is mainly attributable to the low operational costs, the universality and flexibility of detection, as well as the specificity and speed of analysis. Based on characteristic protein spectra obtained from intact cells – by means of simple, rapid and reproducible preanalytical and analytical protocols – MALDI-TOF MS allows a highly discriminatory identification of yeasts and filamentous fungi starting from colonies. Whenever used early, direct identification of yeasts from positive blood cultures has the potential to greatly shorten turnaround times and to improve laboratory diagnosis of fungemia. More recently, but still at an infancy stage, MALDI-TOF MS is used to perform strain typing and to determine antifungal drug susceptibility. In this article, the authors discuss how the MALDI-TOF MS technology is destined to become a powerful tool for routine mycological diagnostics.     

Effect of Culture Conditions on Microorganism Identification by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify bacteria based upon protein signatures. This research shows that while some different proteins are produced by vegetative bacteria when they are cultured in different growth media, positive identification with MALDI-TOF MS is still possible with the protocol established at the Pacific Northwest National Laboratory (K. H. Jarman, S. T. Cebula, A. J. Saenz, C. E. Petersen, N. B. Valentine, M. T. Kingsley, and K. L. Wahl, Anal. Chem. 72:1217-1223, 2000). A core set of small proteins remain constant under at least four different culture media conditions and blood agar plates, including minimal medium M9, rich media, tryptic soy broth (TSB) or Luria-Bertani (LB) broth, and blood agar plates, such that analysis of the intact cells by matrix-assisted laser desorption/ionization mass spectrometry allows for consistent identification.     

Phosphorylation of polyprenols by phosphorous acid amides

Phosphorylation of the hydroxyl group of polyprenol by amides of phosphorous acid with the purpose of producing novel bioactive compounds has been investigated. Di- and monoamidophosphites and thiophosphates of polyprenol have been obtained. Their structure has been confirmed by ³¹P-, ¹H-, and ¹³C NMR spectroscopy and MALDI-TOF mass spectrometry.     

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.     

Structural Characterization and Chemical Classification of Some Bryophytes Found in Latvia

Bryophytes are the second largest taxonomic group in the plant kingdom; yet, studies conducted to better understand their chemical composition are rare. The aim of this study was to characterize the chemical composition of bryophytes common in Northern Europe by using elemental, spectral, and non‐destructive analytical methods, such as Fourier transform IR spectrometry (FT‐IR), solid‐phase ¹³C‐NMR spectrometry, and pyrolysis‐gas chromatography/mass spectrometry (Py‐GC/MS), for the purpose of investigating their chemotaxonomic relationships on the basis of chemical‐composition data. The results of all these analyses showed that bryophytes consist mainly of carbohydrates. Judging by FT‐IR spectra, the OH groups in combination of C? O groups were the most abundant groups. The ¹³C‐NMR spectra provided information on the presence of such compounds as phenolics and lipids. It was found that the amount of phenolic compounds in bryophytes is relatively small. This finding definitely confirmed the absence of lignin in the studied bryophytes. Cluster analysis was used to better understand differences in the chemical composition of bryophyte samples and to evaluate possible usage of these methods in the chemotaxonomy of bryophytes.     

Limits for the detection of (poly-)phosphoinositides by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently established as a powerful tool for the analysis of biomolecules. Here, MALDI-TOF MS was used for the detection of (poly-)phosphoinositides (PPI). PPI possess higher molecular weights than other phospholipids and a high phosphorylation-dependent negative charge. Both features affect the MALDI detection limits expressed as the minimum of analyte on the sample plate resulting in a signal-to-noise-ratio of S/N=5. Using 2,5-dihydroxybenzoic acid (DHB) as matrix the detection limit for phosphatidylinositol (PI) is seven times higher than for phosphatidylcholine (PC) and further increases with increasing phosphorylation or in mixtures with other well-detectable phospholipids. For phosphatidylinositol-tris-phosphate (PIP₃) in a mixture with PC, the limit is about 20 times higher than for PI. The consequences for the experimental conditions are discussed. It is advisable to pre-separate PPI from biological lipid mixtures prior to the application of MALDI-TOF MS.     

Applications of nuclear magnetic resonance in lipid analyses: An emerging powerful tool for lipidomics studies

The role of lipids in cell, tissue, and organ physiology is crucial; as many diseases, including cancer, diabetes, neurodegenerative, and infectious diseases, are closely related to absorption and metabolism of lipids. Mass spectrometry (MS) based methods are the most developed powerful tools to study the synthetic pathways and metabolic networks of cellular lipids in biological systems; leading to the birth of an emerging subject lipidomics, which has been extensively reviewed. Nuclear magnetic resonance (NMR), another powerful analytical tool, which allows the visualization of single atoms and molecules, is receiving increasing attention in lipidomics analyses. However, very little work focusing on lipidomic studies using NMR has been critically reviewed. This paper presents a first comprehensive summary of application of ¹H, ¹³C & ³¹P NMR in lipids and lipidomics analyses. The scientific basis, principles and characteristic diagnostic peaks assigned to specific atoms/molecular structures of lipids are presented. Applications of 2D NMR in mapping and monitoring of the components and their changes in complex lipids systems, as well as alteration of lipid profiling over disease development are also reviewed. The applications of NMR lipidomics in diseases diagnosis and food adulteration are exemplified.     

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape^TM from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of mycobacteria in routine clinical practice

Background

Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed.

Methodology

We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 10⁵ colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25 M. avium and 12 non-tuberculosis clinical isolates with identification scores ≥2 within 2.5 hours.

Conclusions

Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heat-inactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories in both developed and developing countries.