Matrix-assisted Laser Desorption/ionization Mass Spectrometric Research Articles

MALDI mass Spectrometry based proteomics for drug discovery & development

Matrix-assisted laser desorption/ ionization (MALDI) is a soft ionization technique for introducing wide range of analytes into a mass spectrometer (MS). MALDI MS is a powerful tool in drug discovery research and development, providing a high-throughput molecular analysis technique in both preclinical and clinical systems. In particular, MALDI MS is invaluable in the study of peptides and proteins that drive all biological functions. This technology is label-free, provides high specificity in molecular identification, and is high-throughput. MALDI MS has been used in biomarker discovery and quantitation in virtually all tissues, serum, plasma, CSF, and urine for diagnostics, patient stratification, and monitoring drug efficacy. Other applications include characterization of biological drugs, spatial mapping of biomarkers and drugs in tissues, drug screening, and toxicological assessment.

Rapid Detection of Viral Envelope Lipids Using Lithium Adducts and AP-MALDI High-Resolution Mass Spectrometry

There is an unmet need to develop analytical strategies that not only characterize the lipid composition of the viral envelope but also do so on a time scale that would allow for high-throughput analysis. With that in mind, we report the use of atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) combined with lithium adduct consolidation to profile total lipid extracts rapidly and confidently from enveloped viruses. The use of AP-MALDI reduced the dependency of using a dedicated MALDI mass spectrometer and allowed for interfacing the MALDI source to a mass spectrometer with the desired features, which included high mass resolving power (>100000) and tandem mass spectrometry. AP-MALDI combined with an optimized MALDI matrix system, featuring 2',4',6'-trihydroxyacetophenone spiked with lithium salt, resulted in a robust and high-throughput lipid detection platform, specifically geared to sphingolipid detection. Application of the developed workflow included the structural characterization of prominent sphingolipids and detection of over 130 lipid structures from Influenza A virions. Overall, we demonstrate a high-throughput workflow for the detection and structural characterization of total lipid extracts from enveloped viruses using AP-MALDI HRMS and lithium adduct consolidation.

Toxicological alterations induced by subacute exposure of silver nanoparticles in Wistar rats.

Silver nanoparticles (AgNPs) have become crucial players in the field of medicine and various other industries. AgNPs have a wide array of applications, which includes production of electronic goods, cosmetics, synthesis of dyes, and printing inks, as well as targeted delivery of drugs to specialized cells inside the body. Even though humans readily come in contact with these particles, the organ-specific accumulation and resulting mechanisms of toxicity induced by inhaled AgNPs are still under investigation. The goal of this study was to determine the organ distribution of inhaled AgNPs and investigate the resulting systemic toxicity. To do this, male Wistar rats were exposed by inhalation to AgNPs for 4hr/day (200 parts per billion/day) for five consecutive days. The nanoparticles were generated using a laser ablation technique using a soft-landing ion mobility (SLIM) instrument. Inductively coupled plasma mass spectrometric (ICP-MS) analysis showed organ-specific accumulation of the nanoparticles, with the highest concentration present in the lungs, followed by the liver and kidneys. Nanoparticle distribution was characterized in the organs using scanning electron microscopy (SEM) and matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) imaging. Bone marrow cytotoxicity assay of the cells from the femur of rats showed micronuclei formation and signs of cellular cytotoxicity. Moreover, rats displayed increased levels of circulating lactate and glutathione disulphide (GSSG), as determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Collectively, our observations suggest that inhaled subacute exposure to AgNP results in accumulation of AgNPs in the lungs, liver, and kidneys, preferentially, as well as mediates induced systemic toxicity.

Use of NeoSickle Solution in MALDI Mass Spectrometer for the Detection of Hb E and Thalassemia

The NeoSickle® solution based on the detection by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of α-, β- and γ-globin is used in routine clinical practice to classify ...

Fabrication of surface modified ZnO nanorod array for MALDI-MS analysis of bacteria in a nanoliter droplet: a multiple function biochip

We demonstrated the use of surface modified zinc oxide nanorod (ZnO NR) array chip for on-plate detection, pre-concentration, separation and subsequently matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis for bacteria present in nanoliter liquid droplet. The surface modified ZnO NR array can be easily fabricated on Zn foil via chemical method. To explore the potential of the fabricated ZnO NR array chip as a MALDI-MS chip and substrate, standard proteins were studied. It was observed that surface modified ZnO NR array substrates provides significant enhancement on analyte ionization in comparison to the conventional MALDI target plate and efficiently suppress the background noises. This significantly leads to clean mass spectra with high resolution and excellent signal intensity. Furthermore, the proposed method was demonstrated for the analysis of bacteria in nanoliter droplets with high reproducibility and it can be further employed as an effective affinity probe to detect and separate the bacteria from sea water and urine. The lowest detectable concentration of the bacteria cells is 1.9 × 102 cfu μL−1 and the minimum volume required for the sample analysis is 50 nano-liter (nL). We believe that this multiple function biochip can serve as an effective bacteria detecting platform in the near future.

1,6-Diphenyl-1,3,5-hexatriene (DPH) as a Novel Matrix for MALDI MS Imaging of Fatty Acids, Phospholipids, and Sulfatides in Brain Tissues.

1,6-Diphenyl-1,3,5-hexatriene (DPH) is a commonly used fluorescence probe for studying cell membrane-lipids due to its affinity toward the acyl chains in the phospholipid bilayers. In this work, we investigated its use in matrix-assisted laser desorption/ionization (MALDI) as a new matrix for mass spectrometry imaging (MSI) of mouse and rat brain tissue. DPH exhibits very minimal matrix-induced background signals for the analysis of small molecules (below m/z of 1000). In the negative ion mode, DPH permits the highly sensitive detection of small fatty acids (m/z 200-350) as well as a variety of large lipids up to m/z of 1000, including lyso-phospholipid, phosphatidic acid (PA), phosphoethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and sulfatides (ST). The analytes were mostly detected as the deprotonated ion [M - H]-. Our results also demonstrate that sublimated DPH is stable for at least 24 h under the vacuum of our MALDI mass spectrometer. The ability to apply DPH via sublimation coupled with its low volatility allows us to perform tissue imaging of the above analytes at high spatial resolution. The degree of lipid fragmentation was determined experimentally at varying laser intensities. The results illustrated that the use of relatively low laser energy is important to minimize the artificially generated fatty acid signals. On the other hand, the lipid fragmentation obtained at higher laser energies provided tandem MS information useful for lipid structure elucidation.

Novel workflow for combining Raman spectroscopy and MALDI-MSI for tissue based studies.

Molecular heterogeneity of cancer is a major obstacle in tumor diagnosis and treatment. To deal with this heterogeneity, a multidisciplinary combination of different analysis techniques is of urgent need because a combination enables the creation of a multimodal image of a tumor. Here, we develop a computational workflow in order to combine matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) imaging and Raman microspectroscopic imaging for tissue based studies. The computational workflow can be used to confirm a spectral histopathology (SHP) based on one technique with another technique. In this contribution, we confirmed a Raman spectroscopic based SHP with MALDI-imaging. Owing to this combination, we could demonstrate, for a larynx carcinoma sample, that tissue types and different metabolic states could be extracted from the Raman spectra. Further investigations with the help of MALDI spectra yield a better characterization of variable epithelial differentiation and a better understanding of ongoing dysplastic alterations.

Acoustic Sample Deposition MALDI-MS (ASD-MALDI-MS): A Novel Process Flow for Quality Control Screening of Compound Libraries.

In the early stages of drug discovery, high-throughput screening (HTS) of compound libraries against pharmaceutical targets is a common method to identify potential lead molecules. For these HTS campaigns to be efficient and successful, continuous quality control of the compound collection is necessary and crucial. However, the large number of compound samples and the limited sample amount pose unique challenges. Presented here is a proof-of-concept study for a novel process flow for the quality control screening of small-molecule compound libraries that consumes only minimal amounts of samples and affords compound-specific molecular data. This process employs an acoustic sample deposition (ASD) technique for the offline sample preparation by depositing nanoliter volumes in an array format onto microscope glass slides followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. An initial study of a 384-compound array employing the ASD-MALDI-MS workflow resulted in a 75% first-pass positive identification rate with an analysis time of <1 s per sample.

Characterizing changes in snow crab (Chionoecetes opilio) cryptocyanin protein during molting using matrix‐assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry

We report the matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) characterization of the cryptocyanin proteins of the juvenile Chionoecetes opilio crabs during their molting and non-molting phases. In order to assess the structural cryptocyanin protein differences between the molting and non-molting phases, the obtained peptides were sequenced by MALDI low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS). The cryptocyanin protein was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by MALDI-TOF/TOF-MS. The purified cryptocyanin protein was sequenced, using the 'bottom-up' approach. After tryptic digestion, the peptide mixture was analyzed by MALDI-QqTOF-MS/MS and the data obtained were used for the peptide mass fingerprinting (PMF) identification by means of the Mascot database. It was demonstrated using MALDI-TOF/TOF-MS that the actual molecular weights of the non-molting and molting cryptocyanin proteins were different; these were, respectively, 67.6 kDa and 68.1 kDa. Using low-energy CID-MS/MS we have sequenced the trytic peptides to monitor the differences and similarities between the cryptocyanin molecular structures during the molting and non-molting stages. We have demonstrated for the first time that the actual molecular masses of the cryptocyanin protein during the molting and non-molting phases were different. The MALDI-CID-MS/MS analyses allowed the sequencing of the cryptocyanins after tryptic digestion, during the molting and non-molting stages, and showed some similarities and staggering differences between the identified cryptocyanin peptides.

A novel tantalum-based sol–gel packed microextraction syringe for highly specific enrichment of phosphopeptides in MALDI-MS applications

A new tantalum-based sol-gel material was synthesized using a unique sol-gel synthesis pathway by PEG incorporation into the sol-gel structure without performing a calcination step. This improved its chemical and physical properties for the high capacity and selective enrichment of phosphopeptides from protein digests in complex biological media. The specificity of the tantalum-based sol-gel material for phosphopeptides was evaluated and compared with tantalum(V) oxide (Ta2O5) in different phosphopeptide enrichment applications. The tantalum-based sol-gel and tantalum(V) oxide were characterized in detail using FT-IR spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM), and also using a surface area and pore size analyzer. In the characterization studies, the surface morphology, pore volume, crystallinity of the materials and PEG incorporation into the sol-gel structure to produce a more hydrophilic material were successfully demonstrated. The X-ray diffractograms of the two different materials were compared and it was noted that the broad signals of the tantalum-based sol-gel clearly represented the amorphous structure of the sol-gel material, which was more likely to create enough surface area and to provide more accessible tantalum atoms for phosphopeptides to be easily adsorbed when compared with the neat and more crystalline structure of Ta2O5. Therefore, the phosphopeptide enrichment performance of the tantalum-based sol-gels was found to be remarkably higher than the more crystalline Ta2O5 in our studies. Phosphopeptides at femtomole levels could be selectively enriched using the tantalum-based sol-gel and detected with a higher signal-to-noise ratio by matrix-assisted laser desorption/ionization-mass spectrometer (MALDI-MS). Moreover, phosphopeptides in a tryptic digest of non-fat bovine milk as a complex real-world biological sample were retained with higher yield using a tantalum-based sol-gel. Additionally, the sol-gel material was packed into a standard syringe (0.5 mL) to enhance the ease of use of the sol-gel material and for the elimination of additional mixing and separation procedures during the adsorption, washing and elution steps of the enrichment procedure. It was found that up to 28 phosphopeptides in milk digest were easily detectable by MALDI-MS at femtomole levels (around 20 fmol) using the microextraction syringe within less than one minute.

Novel co-matrix systems for the MALDI-MS analysis of polystyrene using a UV absorber and stabilizer

Co-matrix systems composed of a UV absorber and a UV stabilizer applied to the analysis of polystyrene (PS) using a matrix-assisted laser desorption/ionization-mass spectrometer (MALDI-MS) equipped with a N(2) laser (337 nm) as the light source were investigated. Various UV absorbers such as Tinuvin 234, Tinuvin 326, Tinuvin 327, Tinuvin 328, Tinuvin 329, and Tinuvin 1577 and a UV stabilizer of Tinuvin 770 were used. Analytical results using the co-matrix systems were compared with those using anthracene-1,8,9-triol (DIT) as the most common matrix for PS analysis. The MALDI mass spectrum of PS obtained using single DIT showed higher ionization efficiency than that using a mixture of DIT and Tinuvin 770 as the matrix. In contrast to this, for the UV absorbers, the ionization efficiencies were notably enhanced by using the co-matrix systems of the UV absorber and Tinuvin 770. The analytical results were explained with the enhanced compatibility with PS by the formation of a complex between the UV absorber and Tinuvin 770 and with the capability of transferring silver cations. Prices of Tinuvin 770 and the UV absorbers are much cheaper than those of the common matrices. The co-matrix systems are recommendable for the MALDI-MS analysis of PS, especially the co-matrix system composed of Tinuvin 234 and Tinuvin 770.

Abstract 380: Myocardial Angiotensin Metabolism Analysis Using Matrix-Assisted Laser Desorption Ionization (MALDI) Imaging

Mass spectrometric (MS) methods were used to study angiotensin (Ang) metabolism in mouse heart. The focus was on Ang II, Ang-(1-7) and Ang III. Goals were: 1) develop and optimize new technique for in situ assessment of Ang II processing within the myocardium using matrix-assisted laser desorption ionization (MALDI) imaging and 2) to validate results by in vitro MALDI MS enzyme assay as well as specific enzyme inhibitors. This newly developed imaging method has the advantage of allowing for regional spatial visualization and quantification of the formed peptides within the myocardium. Frozen mouse heart sections (12 μm) were incubated with 5 μL of 10-1000 ng/μL Ang II for 5-15 min at 37 °C. The main formed peptides, Ang III and Ang-(1-7), were identified by MALDI-TOF/TOF. Ang-(1-7) was concentrated in the ventricle while Ang III was detected throughout the heart. For the MALDI MS enzyme assay, left ventricular (LV) homogenate was incubated with Ang II at 37 °C. The reaction mixture was spotted on target plate and peptides were detected using MS. Results showed that Ang peptide formation was greatest at 100 ng/μL Ang II with short incubation period. Ang III formation was predominant with Ang III/ Ang II peak intensity 63%, whereas Ang (1-7)/ Ang II peak intensity was 22%. Ang III generation was blocked by the APA inhibitor, glutamate phosphonate (Glu-P), in a concentration dependent manner. The ACE2 inhibitor, MLN-4760, and the PCP/PEP inhibitor, Z-pro-prolinal, reduced myocardial Ang (1-7) formation by 66% (from 70.02 to 23.8 and from 112.7 to 38.3), respectively. Interestingly, blockade of APA by Glu-P caused a 4 fold increase in Ang (1-7)/Ang II ratio. Results validated MALDI imaging for the in situ assessment of Ang II metabolizing enzymes. Ang II is mainly metabolized by APA in the myocardium while ACE2 is the main enzyme responsible for Ang-(1-7) formation from Ang II in the LV. This is the first report on localization of Ang II processing enzymes in the heart using MALDI imaging. This procedure can be exploited for the development of new therapeutic avenues that can be used to target the devastating Ang II effects within the myocardium.

Measurement of laser activated electron tunneling from semiconductor zinc oxide to adsorbed organic molecules by a matrix assisted laser desorption ionization mass spectrometer

Measurement of light induced heterogeneous electron transfer is important for understanding of fundamental processes involved in chemistry, physics and biology, which is still challenging by current techniques. Laser activated electron tunneling (LAET) from semiconductor metal oxides was observed and characterized by a MALDI (matrix assisted laser desorption ionization) mass spectrometer in this work. Nanoparticles of ZnO were placed on a MALDI sample plate. Free fatty acids and derivatives were used as models of organic compounds and directly deposited on the surface of ZnO nanoparticles. Irradiation of UV laser (λ=355nm) with energy more than the band gap of ZnO produces ions that can be detected in negative mode. When TiO2 nanoparticles with similar band gap but much lower electron mobility were used, these ions were not observed unless the voltage on the sample plate was increased. The experimental results indicate that laser induced electron tunneling is dependent on the electron mobility and the strength of the electric field. Capture of low energy electrons by charge-deficient atoms of adsorbed organic molecules causes unpaired electron-directed cleavages of chemical bonds in a nonergodic pathway. In positive detection mode, electron tunneling cannot be observed due to the reverse moving direction of electrons. It should be able to expect that laser desorption ionization mass spectrometry is a new technique capable of probing the dynamics of electron tunneling. LAET offers advantages as a new ionization dissociation method for mass spectrometry.

Extraction of Substance P by Functionalized Iron Oxide Nanoparticles for Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometric Analysis

AbstractFunctionalized iron oxide nanoparticles (IONPs) were used as concentrating probes to extract substance P (SP) from aqueous solution before matrix‐assisted laser desorption/ionization mass spectrometric (MALDI/MS) analysis. Adsorption of SP onto the nanoparticles was mainly due to electrostatic interaction. After simple washing, the SP‐adsorbed nanoparticles were directly analyzed by MALDI/MS. With this concentration strategy, the limit of detection (LOD) of SP in 500 μL buffer solution (pH 8) was 0.1 nM. In addition, the nanoparticles could isolate SP from a solution with high contents of salts and surfactants, thus alleviating suppression effect during MALDI/MS analysis. This new method was successfully applied to the determination of SP in rat spinal cord samples. The LOD and recovery of SP in a rat spinal cord sample were 3.1 nM and 85.7%, respectively.

Multifunctional nanoparticles composite for MALDI-MS: Cd2+-doped carbon nanotubes with CdS nanoparticles as the matrix, preconcentrating and accelerating probes of microwave enzymatic digestion of peptides and proteins for direct MALDI-MS analysis

For the first time, we utilized multifunctional nanoparticles composite (NPs composite) for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of peptides and proteins. Multiwalled carbon nanotubes doped with Cd(2+) ions and modified with cadmium sulfide NPs were synthesized by a chemical reduction method at room temperature. The multifunctional NPs composite applied for the analysis of peptides and microwave-digested proteins in the atmospheric pressure matrix-assisted laser desorption/ionization ion-trap and MALDI time-of-flight (TOF) mass spectrometry (MS) was successfully demonstrated. The maximum detection sensitivity for peptides in MALDI-MS was achieved by the adsorption of negatively charged peptides onto the surfaces of NP composite through electrostatic interactions. The optimal conditions of peptide mixtures were obtained at 20 min of incubation time using 1 mg of NPs composite when the pH of the sample solution was kept higher than the pI values of peptides. The potentiality of the NP composite in the preconcentration of peptides was compared with that of the individual NP by calculating the preconcentration factors (PF) and found that the NPs composite showed a 4-6 times of PF than the other NPs. In addition, the NPs composite was also applied as heat-absorbing materials for efficient microwave tryptic digestion of cytochrome c and lysozyme from milk protein in MALDI-TOF-MS analysis. We believe that the use of NPs composite technique would be an efficient and powerful preconcentrating tool for MALDI-MS for the study of proteome research.

Dansyl‐peptides matrix‐assisted laser desorption/ionization mass spectrometric (MALDI‐MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI‐MS/MS analysis of the proteome

Peptide tagging is a useful tool to improve matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) analysis. We present a new application of the use of the dansyl chloride (DNS-Cl). DNS-Cl is a specific primary amine reagent widely used in protein biochemistry. It adds a fluorescent dimethylaminonaphthalene moiety to the molecule. The evaluation of MALDI-MS and MS/MS analyses of dansylated peptides shows that dansylation raises the ionization efficiency of the most hydrophilic species compared with the most hydrophobic ones. Consequently, higher Mascot scores and protein sequence coverage are obtained by combining MS and MS/MS data of native and tagged samples. The N-terminal DNS-Cl sulfonation improves the peptide fragmentation and promotes the generation of b-fragments allowing better peptide sequencing. In addition, we set up a labeling protocol based on the microwave chemistry. Peptide dansylation proved to be a rapid and cheap method to improve the performance of liquid chromatography (LC)/MALDI-MS/MS analysis at the proteomic scale in terms of peptide detection and sequence coverage.

Effect of Gas Pressure and Gas Type on the Fragmentation of Peptide and Oligosaccharide Ions Generated in an Elevated Pressure UV/IR-MALDI Ion Source Coupled to an Orthogonal Time-of-Flight Mass Spectrometer

Matrix-assisted laser desorption ionization (MALDI) allows for the mass spectrometric (MS) analysis of thermally labile, non-volatile biomolecules. However, some residual analyte fragmentation typically accompanies the phase transition from the condensed to the gas phase and following plume expansion, even under optimized conditions. In-source decay (ISD) and post-source decay (PSD) MALDI MS are two techniques that make use of these phenomena and that can provide useful structural information by producing characteristic fragment ions of the analyte compounds. In orthogonal extracting time-of-flight mass spectrometry (o-TOF-MS), the pressure of the cooling gas in the ion source has a strong influence on the extent of analyte ion fragmentation. We investigated the effect of this parameter on peptide and oligosaccharide fragmentation by examining a range of pressures (from 0.05-1.8 mbar) in combination with seven different buffer gases (He, Ne, Ar, N(2), CO(2), CH(3), isobutane). Ions were generated by ultraviolet (UV) and/or by infrared (IR) MALDI. The influence of the ion extraction voltage on the analyte fragmentation also was investigated for a selected set of gas parameters. We observed that individual fragment ions exhibit characteristic fragment yield-pressure dependencies that can be classified into three groups. Type I ions resemble species that are also found in MALDI PSD MS analysis, while type II ions resemble typical ISD fragments. The yield-pressure relationship of type III ions suggests that these are the result of a combination of both processes. Comparing the yields of fragmentation for the different buffer gases reveals a correlation between their internal degrees of freedom and their collisional cooling efficiency. Changing the buffer gas pressure and/or extraction field provides an easy means to influence analyte ion fragmentation and to switch from the primary production of one type of fragment species to another. The method can therefore facilitate the structural characterization of MALDI-generated ions.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Polysulfated-Derived Oligosaccharides Using Pyrenemethylguanidine

A better understanding of the biological roles of carbohydrates requires the use of tools able to provide efficient and rapid structural information. Unfortunately, highly acidic oligomers-such as polysulfated oligosaccharides-are very challenging to characterize because of their high polarity, structural diversity, and sulfate lability. These features pose special problems for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis because polysulfated carbohydrates exhibit poor ionization efficiency and usually do not produce any signal. The present report demonstrates how MALDI-MS can be used to derive structural and compositional information from pure and mixed fractions of polysulfated oligosaccharides. Indeed, pyrenemethylguanidine (pmg, a derivatizing agent and ionization efficiency enhancer) was used for the analysis of di- to decasaccharides, carrying from two to nine sulfate groups. The method is applied to various highly sulfated chondroitin and carrageenan oligosaccharides as well as to the analysis of mixtures of compounds. In the mass spectra, the observation of a unique pmg-complexed ladder of peaks in both ionization modes allows an easy and rapid determination of both the number of sulfate groups carried by the analyte and its molecular weight. Moreover, we have developed a software tool for the rapid and automatic structural elucidation of carrageenans based on the mass spectra obtained.

Lys Tag: an easy and robust chemical modification for improved de novo sequencing with a matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometer

Mass spectrometry using a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) instrument is a widespread technique for various types of proteomic analysis. Along with an expanding interest in proteomics, there is a strong requirement for the identification of proteins with high confidence from biological samples. Peptide modification by a wide variety of post-translational modifications (PTMs), the existence of different protein isoforms and the presence of uncharacterized genomes of many species, make protein identification through peptide mass fingerprinting (PMF) often unachievable. Peptide de novo sequencing has been proven to be a useful approach to overcome these variables, and efficient derivatization processes are important tools to achieve this goal. In the present work we describe the methodology and experimental applications of a fast, efficient and cheap lysine derivatization. This chemical modification improves the signals from lysine-terminated peptides and can be efficiently used as a lysine-blocking agent in combination with other derivatization techniques. Most importantly, upon peptide fragmentation it generates a neat series of predominantly y-ions, allowing the determination of unambiguous amino acid sequences. Moreover, this chemical compound was used with target-eluted samples, enabling a second, alternative analysis of the same sample in the MALDI mass spectrometer.

Transparent triethylamine‐containing MALDI matrices

AbstractIn matrix‐assisted laser desorption/ionization (MALDI) mass spectrometric (MS) imaging of biological tissues, an organic matrix is deposited onto a sample and co‐crystallized with the peptides and proteins that are extracted from the tissue. Thus, analytes are incorporated into matrix crystals, making them accessible to the matrix‐assisted desorption/ionization process. However, crystal dimensions can limit the spatial resolution of these imaging experiments and hinder observation of small features in the tissue. The addition of differing amounts of triethylamine (TEA) to solutions of common MALDI matrices—α‐cyano‐4‐hydroxycinnamic acid (CHCA), 2,5‐dihydroxybenzoic acid, and sinapinic acid—results in the formation of liquid matrices that may alleviate these problems. Liquid matrices form optically transparent, viscous films that do not crystallize, even when kept open to air for several days at room temperature or placed in the vacuum of the mass spectrometer. These matrices allow peptides to be characterized using MALDI MS down to the femtomole level. Although signal intensity is reduced in liquid matrices as compared to standard crystalline matrices, a phenomenon especially evident for higher molecular mass analytes, peptides are still detected even when TEA comprises 2/3 (v/v) of the MALDI matrix mixture. Transparent matrices demonstrate excellent repeatability and sample stability. Post‐source decay sequencing of peptides with a CHCA/TEA matrix produces intense peptide fragment signals in the low molecular mass region, and rat pituitary blots yield high‐quality spectra using these mixtures. Overall, TEA‐based transparent MALDI matrices are promising for coupling MALDI MS and light microscopy.