Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometry

Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid.     

Mass spectrometric analysis of low molecular mass polyesters by laser desorption/ionization on porous silicon

A low molecular mass polyester was analyzed by desorption/ionization on porous silicon (DIOS) mass spectrometry. The results were compared with those of matrix-assisted laser desorption ionization (MALDI) mass spectrometry using matrixes of alpha-cyano-4-hydroxycinnamic acid (CHCA) and 10,15,20-tetrakis(pentafluorophenyl)porphyrin (F20TPP). The CHCA matrix was not suitable for characterization of low molecular mass components of the polyester because the matrix-related ions interfered with the component ions. On the other hand, the F20TPP matrix showed no interference because no matrix-related ions appeared below m/z 822. However, the solvent selection for determining optimal conditions of sample preparation was limited, because F20TPP does not dissolve readily in any of the available organic solvents. In the DIOS spectra, the polymer ions were observed at high sensitivity without a contaminating ion. No matrix is needed for DIOS spectra of low molecular mass polyesters, facilitating sample preparation and selectivity of a precursor ion in post-source decay measurements.     

Fabrication of nanostructured silicon by metal-assisted etching and its effects on matrix-free laser desorption/ionization mass spectrometry

A matrix-free, high sensitivity, nanostructured silicon surface assisted laser desorption/ionization mass spectrometry (LDI-MS) method fabricated by metal-assisted etching was investigated. Effects of key process parameters, such as etching time, substrate resistance and etchant composition, on the nanostructured silicon formation and its LDI-MS efficiency were studied. The results show that the nanostructured silicon pore depth and size increase with etching time, while MS ion intensity increases with etching time to 300 s then decreases until 600 s for both low resistance (0.001–0.02 Ω cm) and high resistance (1–100 Ω cm) silicon substrates. The nanostructured silicon surface morphologies were found to directly affect the LDI-MS signal ion intensity. By characterizing the nanostructured silicon surface roughness using atomic force microscopy (AFM) and sample absorption efficiency using fluorescence microscopy, it was further demonstrated that the nanostructured silicon surface roughness was highly correlated to the LDI-MS performance.     

Coffee-ring effects in laser desorption/ionization mass spectrometry

This report focuses on the heterogeneous distribution of small molecules (e.g. metabolites) within dry deposits of suspensions and solutions of inorganic and organic compounds with implications for chemical analysis of small molecules by laser desorption/ionization (LDI) mass spectrometry (MS). Taking advantage of the imaging capabilities of a modern mass spectrometer, we have investigated the occurrence of “coffee rings” in matrix-assisted laser desorption/ionization (MALDI) and surface-assisted laser desorption/ionization (SALDI) sample spots. It is seen that the “coffee-ring effect” in MALDI/SALDI samples can be both beneficial and disadvantageous. For example, formation of the coffee rings gives rise to heterogeneous distribution of analytes and matrices, thus compromising analytical performance and reproducibility of the mass spectrometric analysis. On the other hand, the coffee-ring effect can also be advantageous because it enables partial separation of analytes from some of the interfering molecules present in the sample. We report a “hidden coffee-ring effect” where under certain conditions the sample/matrix deposit appears relatively homogeneous when inspected by optical microscopy. Even in such cases, hidden coffee rings can still be found by implementing the MALDI-MS imaging technique. We have also found that to some extent, the coffee-ring effect can be suppressed during SALDI sample preparation.     

Analysis of deprotonated acids with silicon nanoparticle-assisted laser desorption/ ionization mass spectrometry

Chemically modified silicon nanoparticles were applied for the laser desorption/negative ionization of small acids. A series of substituted sulfonic acids and fatty acids was studied. Compared to desorption ionization on porous silicon (DIOS) and other matrix-less laser desorption/ionization techniques, silicon nanoparticle-assisted laser desorption/ionization (SPALDI) mass spectrometry allows for the analysis of acids in the negative ion mode without the observation of multimers or cation adducts. Using SPALDI, detection limits of many acids reached levels down to 50 pmol/μl. SPALDI of fatty acids with unmodified silicon nanoparticles was compared to SPALDI using the fluoroalkyl silylated silicon powder, with the unmodified particles showing better sensitivity for fatty acids, but with more low-mass background due to impurities and surfactants in the untreated silicon powder. The fatty acids exhibited a size-dependent response in both SPALDI and unmodified SPALDI, showing a signal intensity increase with the chain length of the fatty acids (C12-C18), leveling off at chain lengths of C18-C22. The size effect may be due to the crystallization of long chain fatty acids on the silicon. This hypothesis was further explored and supported by SPALDI of several, similar sized, unsaturated fatty acids with various crystallinities. Fatty acids in milk lipids and tick nymph samples were directly detected and their concentration ratios were determined by SPALDI mass spectrometry without complicated and time-consuming purification and esterification required in the traditional analysis of fatty acids by gas chromatography (GC). These results suggest that SPALDI mass spectrometry has the potential application in fast screening for small acids in crude samples with minimal sample preparation.     

Matrix-free infrared soft laser desorption/ionization

Infrared soft laser desorption/ionization was performed using a 2.94 µm Er : YAG laser and a commercial reflectron time-of-flight mass spectrometer. The instrument was modified so that a 337 nm nitrogen laser could be used concurrently with the IR laser to interrogate samples. Matrix-assisted laser desorption/ionization (MALDI), laser desorption/ionization and desorption/ionization on silicon with UV and IR lasers were compared. Various target materials were tested for IR soft desorption ionization, including stainless steel, aluminum, copper, silicon, porous silicon and polyethylene. Silicon surfaces gave the best performance in terms of signal level and low-mass interference. The internal energy resultant of the desorption/ionization was assessed using the easily fragmented vitamin B₁₂ molecule. IR ionization produced more analyte fragmentation than UV-MALDI analysis. Fragmentation from matrix-free IR desorption from silicon was comparable to that from IR-MALDI. The results are interpreted as soft laser desorption and ionization resulting from the absorption of the IR laser energy by the analyte and associated solvent molecules. Copyright © 2004 John Wiley & Sons, Ltd.     

Desorption/ionization on porous silicon mass spectrometry (DIOS) of model cationized fatty acids

Desorption/ionization on porous silicon (DIOS) is a very useful technique in the case of small molecular weight compounds, compared to the matrix-assisted laser desorption ionization (MALDI). This is because MALDI generates matrix-related ions that overlap with the mass range of interest. The aim of our work was to investigate the suitability of the DIOS technique in the case of fatty acids in negative ion mode. The analysis of the chosen fatty acid models, nonadecanoic acid (C(19)H(38)O(2)) and heneicosanoic acid (C(21)H(42)O(2)), gave rise to the observation of the deprotonated monomeric species and selective cationized multimeric species. This cation selectivity was further elucidated by complementary studies based on the addition of various metals such as Ag(I), Zn(II), Fe(II), and also Cu(II). Specific behavior, depending upon the introduced metal, was highlighted by different redox reaction processes and also metastable decompositions (in PSD mode).     

Measurement of serum salicylate levels by solid-phase extraction and desorption/ionization on silicon mass spectrometry

The applicability of the matrix-free laser desorption/ionization on silicon mass spectrometry (DIOS-MS) to measuring serum drug levels was examined by analyzing serum salicylic acid. The optimized and simple solid-phase extraction (SPE) allowed good recovery, 88.9 +/- 5.8%, for 1.4 mM (200 mg/L) of salicylic acid in serum. The negative ion MS allowed measurements of deprotonated molecules without interference from other signals. Using a deuterium-labeled internal standard, good linearity was obtained in the 0.14 to 4.2 mM (20-600 mg/L) range, which was sufficient for monitoring the therapeutic anti-inflammatory dose. SPE followed by DIOS-MS is anticipated to be a method of measuring drug levels in blood and may allow high throughput analysis.     

Analysis of amphetamines and fentanyls by atmospheric pressure desorption/ionization on silicon mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry and its application to forensic analysis of drug seizures

The suitability of atmospheric pressure desorption/ionization on silicon mass spectrometry (AP-DIOS-MS) and matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI-MS) for the identification of amphetamines and fentanyls in forensic samples was studied. With both ionization techniques, the mass spectra recorded showed abundant protonated molecules, and the background did not disturb the analysis. The use of tandem mass spectrometry (MS/MS) allowed unambiguous identification of the amphetamines and fentanyls. AP-DIOS-MS/MS and AP-MALDI-MS/MS were also successfully applied to the identification of authentic compounds from drug seizures. Common diluents and tablet materials did not disturb the analysis and compounds were unequivocally identified. The limits of detection (LODs) for amphetamines and fentanyls with AP-DIOS-MS/MS were 1-3 pmol, indicating excellent sensitivity of the method. The LODs with AP-MALDI-MS/MS were about 5-10 times higher.     

Multiphoton ionization spectroscopy in surface analysis and laser desorption mass spectrometry

The application of resonance-enhanced multiphoton ionization (REMPI) spectroscopy for the ultrasensitive detection of molecules originating from laser desorption experiments performed on a variety of substrates is reviewed. Laser-induced desorption from surfaces is capable of producing intact gas-phase molecules, even from polar, non-volatile, high-molecular-weight and thermally labile substances. REMPI is a highly efficient and optically selective ionization method, which, coupled with laser desorption allows the direct chemical analysis of complex mixtures, without the need for previous sample purification and separation steps. The use of REMPI spectroscopy is discussed in two contexts: (1) for the direct chemical analysis of complex mixtures, e.g., environmental samples, by laser desorption/laser postionization mass spectrometry and (2) for measurements of internal state distribution of molecules laser-desorbed from sub-monolayers surface films to gain insight into the laser desorption mechanism.Presented at the 13th International Symposium on Microchemical Techniques (ISM), held in Montreux, Switzerland, May 16–20,1994     

Matrix-assisted laser desorption ionization mass spectrometry for identification of shrimp

Matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry was used to identify shrimp at the species level using commercial mass spectral fingerprint matching software (Bruker Biotyper). In the first step, a mass spectrum reference database was constructed from the analysis of six commercially important shrimp species: Litopenaeus setiferus, Farfantepenaeus aztecus, Sicyonia brevirostris, Pleoticus robustus, Pandalopsis dispar and Pandalus platyceros. This step required a desalting procedure for optimum performance. In the second step, the reference database was tested using 74 unknown shrimp samples from these six species. Correct identification was achieved for 72 of 74 samples (97%): 72 samples were identified at the species level and 2 samples were identified at the genus level using the manufacturer's log score specifications. The MALDI fingerprinting method for the identification of shrimp species was found to be reproducible and accurate with rapid analysis.     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

等离子体常压解吸离子源质谱

近年来,常压开源质谱解吸/离子化(ambient desorption/ionization mass spectrometry)技术的开发促进了质谱分析的蓬勃发展.作为常压开源质谱技术的一大重要分支,基于等离子体的常压质谱解吸/离子化技术因无需样品预处理、无溶剂化过程、高灵敏度、高通量、能实时在线检测样品等优点,受到了学术界及仪器制造、化学和生物分析等相关产业界的广泛关注.本文从离子源的种类、原理及应用等角度对目前我国在开源质谱领域中自主研发的基于等离子体的常压离子源的研究进展进行了总结,并对此类等离子体常压离子源的发展进行了展望,重点涉及微波等离子体源、常压微辉光放电等离子体源和解吸电晕束离子源.     

基质辅助激光解吸电离质谱和电喷雾电离质谱在辣根过氧化物酶糖肽结构分析中的应用

糖链结构的质谱解析是今后糖蛋白分析中的重要研究内容,其中完整糖肽的分析,由于可以同时获得糖基化位点和对应糖链的结构信息,更具有重要意义和研究前景。本工作对质谱软电离技术在完整糖肽分析中的应用进行了研究,其中包括了基质辅助激光解吸电离(matrix-assisted laser desorption ionization, MALDI)和电喷雾电离(electrospray ionization, ESI)技术。通过平行使用两种串联质谱(tandem mass spectrometry, MS/MS)分析策略: MALDI-MS/MS和ESI-MS/MS对目标糖蛋白——辣根过氧化物酶进行分析,并讨论了其互补性。结果表明,MALDI和ESI技术各有优劣,结合串联质谱分析,可获得糖肽的糖链结构信息;两条路线互补使用,在揭示蛋白质糖基化修饰(位点和结构)的研究中十分必要。     

Miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry

The trend of miniaturization in bioanalytical chemistry is shifting from technical development to practical application. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), progress in miniaturizing sample spots has been driven by the needs to increase sensitivity and speed, to interface with other analytical microtechnologies, and to develop miniaturized instrumentation.We review recent developments in miniaturizing sample spots for MALDI-MS. We cover both target modification and microdispensing technologies, and we emphasize the benefits with respect to sensitivity, throughput and automation.We hope that this review will encourage further method development and application of miniaturized sample spots for MALDI-MS, so as to expand applications in analytical chemistry, protein science and molecular biology.     

Matrix-assisted laser desorption/ionization mass spectrometry analysis of glycans with co-derivatization of asparaginyl-oligosaccharides

As one of the most prevalent and complex post-translational modifications in biological systems, proteins glycosylation has drawn considerable attention in recent decades. Dissociation of the carbohydrates from glycoproteins may be the prerequisite step of glycomics experiments, which commonly performed by specific proteolysis. In this study, an alternative strategy was reported with nonspecific proteolysis in coupling with co-derivatization of TMPP-Ac and methylamidation for glycan moieties analysis by MALDI-MS. With the co-derivatization, a permanent positive charge was introduced to the Asn-glycans and the carboxylic groups were neutralized by methylamidation simultaneously. As a result, approximately 20 and 50-fold enhancement in the detection sensitivity was achieved for asialo-Asn and disialo-Asn respectively in comparison to their native counterparts. Ultimately, this developed strategy was successfully validated using three model glycoproteins, including ribonuclease B, ovalbumin and transferrin.     

Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.     

Studies on extracting solutions of endohedral rare-earth metallofullerenes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Thirteen extracting solutions of rare-earth metallofullerenes containing La,Ce,Pr,Nd Sm,Eu,Gd,Tb,Dy,Ho,Er,Tm and Yb respectively have been investigated by means of matrix-assisted laser desorpuon/ ionization time-of-flight mass spectrometry.The influences of the positive-ion/negative-ion mode,laser intensity,ma trix and mass discrimination to the analytical results are studied,based on which the optimal analytical conditions have been determined.The results show that the extracting solutions contain large quantities of rare-earth metallofullerenes besides empty fullerenes.On the basis of comparing their relative intensities,the different structure stabilities and solubilities of metallofullerenes with different rare-earth metals encapsulated into the fullerene cages,as well as some possible reasons to those differences,are discussed.     

Study of synthetic aliphatic copolyamides by time-of-flight matrix assisted laser desorption/ionization mass spectrometry

Synthetic copolyamides based on aliphatic diamines (1,3-propanediamine and 1,4-butanediamine) and dichlorides of aliphatic carboxylic acids (adipic and sebacic acid dichlorides) were investigated using time-of-flight matrix assisted laser desorption/ionization mass spectrometry. Their mass spectra showed peaks for cationized (Na⁺ and K⁺) and protonated (less intense peaks) oligomers with NH₂-NH₂, NH₂-COOH, or COOH-COOH end groups. No cyclic oligomers were detected in the samples. The compositions of oligomers were determined, and the relative reactivities of homologous comonomers in polycondensation were estimated. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1320–1324, July, 2007.     

Derivatization of small biomolecules for optimized matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a powerful tool for the measurement of low molecular mass compounds of biological interest. The limitations for this method are the volatility of many analytes, possible interference with matrix signals or bad ionization or desorption behavior of the compounds. We investigated the application of well-known and straightforward one-pot derivatization procedures to circumvent these problems. The derivatizations tested allow the measurement and the labeling of alcohols, aldehydes and ketones, carboxylic acids, alpha-ketocarboxylic acids and amines.