Characterization of poly(L-lactide)-block-poly(ethylene oxide)-block-poly(L-lactide) triblock copolymer by liquid chromatography at the critical condition and by MALDI-TOF mass spectrometry

Poly(L-lactide)-block-poly(ethylene oxide)-block-poly(L-lactide) triblock copolymers (PLLA-b-PEO-b-PLLA) were fractionated in terms of the number of LLA units by liquid chromatography at the critical condition (LCCC) of PEO block. The fractionated samples were identified using MALDI-TOF mass spectrometry. The dependence of the LCCC retention of the diblock and triblock copolymers on the degree of polymerization of PLLA block(s) follows Martin's rule very well. Unlike the case of PEO-b-PLLA diblock copolymer reported earlier (Lee, H.; et al. Macromolecules 1999, 32, 4143), however, a splitting of the elution peaks containing the same number of LLA units was found. The peak splitting was ascribed to the different length distributions of PLLA blocks at the two ends of the PEO block. From the relative intensities of the peaks, the split peaks were assigned to different isomeric structures of the PLLA blocks. From these results we conclude that the interaction of the triblock copolymers with the stationary phase is affected by the distribution of the interacting blocks at the two ends of the center PEO block, in addition to the total number of LLA units in the triblock copolymer.     

Analysis of viral glycoproteins by MALDI-TOF mass spectrometry

Membrane glycoproteins were shown to be useful biomarkers of enveloped viruses using on-target deglycosylation and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Sindbis virus, the prototype alpha-virus, was used as a model system. The glycoproteins and the capsid protein of the Sindbis virus were successfully detected by MALDI-TOF MS using two solvent systems. One of them is 0.5% n-octyl glucoside/0.5% trifluoroacetic acid. The two components of this solvent acted synergistically on the virus to help release and solubilize the structural proteins. The other is 70% acetonitrile/30% formic acid. This solvent solubilized the integral membrane glycoproteins very effectively even after serious aggregation. On-target deglycosylation was performed to confirm the detection of the glycoprotein peak and to produce protein moieties that can be used as biomarkers. After a simple and fast incubation using peptide-N-glycosidase F on target, sequential mass shifts were observed, which proved that the proteins detected at 51 000 Da have N-linked carbohydrate moieties at two sites. Observation of this mass shift could provide confirmatory evidence for viral identification.     

PEG改性聚乳酸嵌段共聚物降解性能研究

辛酸亚锡催化下开环聚合制备聚乳酸(DL-PLA)-聚乙二醇(PEG)-聚乳酸三嵌段共聚物.用GPC、DSC、1 H-NMR、质量损失、静态接触角等方法在pH=7.4磷酸盐缓冲液中,37℃下研究了分子量Mn=400、1000和4000的PEG改性DL-PLA的降解行为.结果表明,PEG嵌段增强了共聚物的亲水性,降低了共聚物Tg,加速了共聚物降解,随着PEG分子量增加及两端DL-PLA链段增长,共聚物分子量下降速率加快.由研究结果得出,共聚物降解期间DL-PLA链段中的酯键随机断裂,PEG两端DL-PLA链段逐渐变短;降解后期DL-PLA链段进一步变短,并有短链DL-PLA均聚物产生,当PEG两端DL-PLA链段足够短时,共聚物在介质中溶解.     

Aflatoxin screening by MALDI-TOF mass spectrometry

Efficient detection of aflatoxins B1, B2, G1, and G2 has been performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a UV-absorbing ionic liquid matrix to obtain "matrix-free" mass spectra and addition of NaCl to enhance sensitivity via Na+ cationization. Using ionic alpha-cyano-4-hydroxycinnamic acid (Et3N-alpha-CHCA) as the matrix, matrix-free mass spectra in the m/z range of interest are acquired, and the B1, B2, G1, and G2 aflatoxins are readily detected with an LOD as low as 50 fmol. The technique is fast, requires little sample preparation and no derivatization or chromatographic separation, and seems therefore to be suitable for high-throughput aflatoxin screening. It should be easily extended to other micotoxins and provide an attractive technique to control the quality of major crops subjected to huge world commercial trades such as peanuts, corn, and rice as well as to monitor bioterrorism threats by micotoxin poisoning.     

Temperature gradient interaction chromatography and MALDI-TOF mass spectrometry analysis of stereoregular poly(ethyl methacrylate)s

Temperature gradient interaction chromatography (TGIC) was applied for the separation of stereoregular poly(ethyl methacrylate) (PEMA) according to the tacticity. The three PEMA samples with differing tacticity (rr triad content 0, 53, and 91%) prepared by anionic polymerization were used. C18 bonded silica and a mixture of CH2Cl2 and CH3CN (30/70, v/v) were used as stationary and mobile phase, respectively. TGIC was able to separate the PEMA samples, showing the increasing retention in the order of decreasing rr triad contents; however TGIC elution peaks of the three PEMAs were not fully resolved but, rather, were partially overlapped. To isolate the tacticity effect from the molecular weight effect on the TGIC retention, the PEMA samples were fractionated by TGIC, and the accurate molecular weight of the fractions was determined by MALDI-TOF mass spectrometry. The fractions showed a much narrower molecular weight distribution than the mother PEMAs. The TGIC fractions of similar molecular weight but with different tacticity were fully resolved by TGIC, but mother PEMAs were not. These results indicate that the retention in TGIC is affected by both tacticity and molecular weight.     

Characterization of an insoluble poly(9,9-diphenyl-2,7-fluorene) by solvent-free sample preparation for MALDI-TOF mass spectrometry

The application of solvent-free sample preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allowed the characterization of an insoluble fraction of poly(9,9-diphenylfluorene) that was previously hindered by the lack of suitable characterization methods. The MALDI mass spectrometric analysis gives valuable mechanistic information about the heterogeneous polymerization process of the insoluble high molecular weight fraction of the polymer. The fragmentation appearing even under moderate desorption and ionization conditions of this rigid backbone analyte is identified as a multiple loss of the bulky phenyl side groups and can be avoided by applying the new MALDI matrix 7,7,8,8-tetracyanoquinodimethane. A specialized fragmentation study by postsource decay MALDI-TOF MS reveals a molecular weight dependent change in fragmentation mechanism from an exclusive cleavage of side groups from long polymer chains to an additional cleavage of the polymer backbone of short polymer chains.     

聚乙二醇改性聚乳酸嵌段共聚物的合成与亲水性研究

以DL-丙交酯和分子量Mn=400、1000和2000聚乙二醇(PEG)为原料,在辛酸亚锡催化下开环聚合制备了聚乳酸(PLA)-聚乙二醇-聚乳酸三嵌段共聚物(PLEG)。考察了催化剂用量、反应时间对产率和[η]的影响。用FT-IR、1 H-NMR、GPC、DSC、XRD、静态水接触角等对共聚物进行了表征和性能测试。结果表明,催化剂用量为0.2%、反应时间分别为PLEG400共聚物2～4h、PLEG1000共聚物4～8h和PLEG2000共聚物8～12h较宜;共聚物组成比与投料比较一致,共聚物的数均分子量与理论计算值较一致;共聚物为无定形态,PEG的引入使共聚物Tg明显低于PLA均聚物,且随PEG的Mn减小,共聚物的Tg随之降低;而且PEG的引入明显提高了PLA的亲水性,PEG的Mn越小,PLA亲水性的提高程度越大。通过控制催化剂用量和反应时间,150℃可以得到分子量符合投料组成比要求、亲水性有明显提高的PLA-PEG-PLA三嵌段共聚物。     

Bioinert membranes prepared from amphiphilic poly(vinyl chloride)-g-poly(oxyethylene methacrylate) graft copolymers

Poly(vinyl chloride) (PVC) membrane was hydrophilically modified by grafting with poly(oxyethylene methacrylate) (POEM) using atom transfer radical polymerization (ATRP). The successful grafting of PVC main chain by POEM was characterized by Fourier transform infrared spectroscopy (FT-IR). The molecular weight and hydrophilicity of membranes increased with the amount of POEM grafting, as characterized by gel permeation chromatography (GPC) and contact angle measurement, respectively. Transmission electron microscope (TEM) and small angle X-ray scattering (SAXS) analysis revealed the microphase-separated structure of PVC-g-POEM and the domain spacing increased from 59.3 to 86.1 nm with increasing grafting degree. Scanning electron microscopy (SEM) was used for the direct visualization of the mouse embryonic fibroblast (MEF) cell and bacteria adhesion on the membrane surface. Protein adsorption and eukaryotic and prokaryotic cell adhesion tests showed that the bioinert properties of membranes were significantly increased with POEM content.     

Determination of Block Length Distributions of Poly(oxypropylene) and Poly(oxyethylene) Block Copolymers by MALDI-FTICR Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) was performed on an external ion source Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to analyze the block length distributions of triblock polymers of poly(oxypropylene) and poly(oxyethylene). The first series of results presented demonstrate that the apparent molecular weight distributions are distorted. This distortion is induced by the flight-time-induced mass discrimination inherent in the experimental technique, the variation of isotopic patterns over the measured mass range, and the overlap of peaks in the spectrum. Subsequently, a method for the treatment of molecular weight distributions measured by MALDI on an external ion source FTICR-MS is developed to yield the actual molecular weight distribution and, from that, the individual block length distributions. For the first time, detailed and accurate molecular weight data were obtained on a complex sample using this methodology, which independently validates the data provided by the manufacturer. The experimentally verified random coupling hypothesis proves the validity of the methodology.     

Accurate molecular mass determination of mycolic acids by MALDI-TOF mass spectrometry

Mycolic acids, major and specific long-chain fatty (C70-C90) acid components of the mycobacterial cell envelope, were analyzed for the first time using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry operating in a reflectron mode. The various types of purified mycolates from representative mycobacterial species were analyzed using 2,5-DHB as matrix, because less than 10 pmol of mycolates was sufficient to obtain well-resolved mass spectra composed exclusively of pseudomolecular [M + Na]+ ions consistent with the structures deduced from the chemical analytical techniques applied to these molecules. Examination of the MALDI mass spectra demonstrated that the chain lengths of the various mycolates correlated with the growth rate of mycobacterial strains. Although slow growers, such as Mycobacterium tuberculosis and Mycobacterium ulcerans, produced a series of odd carbon numbers (C74-C82) of alpha-mycolic acids, rapid growers synthesized both odd and even carbon numbers. In addition, the main chain of oxygenated mycolic acids from slow growers were four to six carbon atoms longer than the corresponding alpha-mycolic acids, whereas rapid growers elaborated oxygenated homologues possessing the same chain lengths as their alpha-mycolic acids. Furthermore, a comparative analysis of the crude fatty acid mixtures from a wild-type strain of M. tuberculosis and its isogenic mutant effected in the synthesis of oxygenated mycolates by MALDI mass spectrometry revealed structural differences between the alpha-mycolates from the two strains. Thus, this technique appeared to be a rapid and highly sensitive technique for the analysis of mycolic acids, not only by providing accurate molecular masses and new structural information, but also by both reducing sample consumption and saving time.     

聚醚聚硅氧烷两亲性三嵌段共聚物的合成研究

以Si-H双封端硅油和烯丙基聚醚为原料,通过硅氢加成,进行无溶剂体系PEO-b- PDMS-b- PEO共聚物的催化合成.系统考察了氯铂酸分子中胺类分子的掺杂种类及用量、反应时间和反应温度对反应体系Si-H转化率的影响.结果表明:当Bu3N以n(N)/n(Pt)＝1的量对H2 PtCl6进行掺杂,且反应温度为90℃及反...     

Oligosaccharide structures studied by hydrogen-deuterium exchange and MALDI-TOF mass spectrometry

Hydrogen-deuterium exchange matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (HX-MALDI-TOF MS) is reported for the determination of exchangeable protons in diverse oligosaccharide and glycoconjugate structures. The method has broad application for determining carbohydrate structure and conformation and to the study of carbohydrate-ligand interactions. The proton exchange process has been optimized to maximize the forward deuterium exchange and to suppress the well-known problem of back-exchange and is suitable for the analysis of all exchangeable proton types in carbohydrates. This has been validated for several diverse carbohydrate structures, including series of malto- and xylopyranose oligosaccharides; alpha- and beta-cyclodextrins; a nonreducing tetrasaccharide, stachyose; an N-acetylamide-containing oligosaccharide, chitotetraose; and a tertiary hydroxyl-containing antibiotic glycoconjugate, erythromycin.     

Depth profiling of poly(L-lactic acid)/triblock copolymer blends with time-of-flight secondary ion mass spectrometry

Time-of-flight secondary ion mass spectrometry employing an SF5+ polyatomic primary ion source was utilized to obtain a series of in-depth profiles from PLLA/Pluronic-P104 (poly(ethylene oxide-co-propylene oxide) triblock copolymer) blends in attempts to quantify the in-depth surface segregated Pluronic region. The resultant in-depth profiles were consistent with theoretical models describing the surface segregated region in polymeric blends and copolymer systems, with a surface enriched Pluronic-P104 region, followed by a P104 depletion layer, and finally a constant composition bulk region. These results were consistent over a range of concentrations (1-25%). The depth profiles obtained using cluster SIMS were compared to information obtained using X-ray photoelectron spectroscopy. The results demonstrate that, with cluster primary ion bombardment, we are for the first time able to quantify the polymeric composition as a function of depth within certain multicomponent polymer blends. This success can be attributed to the sputter characteristics of polyatomic primary ion bombardment (SF5+) as compared to monatomic primary ion beams.     

Characterization of Enterobacteria using MALDI-TOF mass spectrometry

A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order.     

Qualitative and quantitative analysis of pharmaceutical compounds by MALDI-TOF mass spectrometry

In this report, we discuss key issues for the successful application of MALDI-TOF mass spectrometry to quantify drugs. These include choice and preparation of matrix, nature of cationization agent, automation, and data analysis procedures. The high molecular weight matrix meso-tetrakis(pentafluorophenyl)porphyrin eliminates chemical noise in the low-mass range, a "brushing" spotting technique in combination with prestructured target plates enables fast preparation of homogeneous matrix crystals, and addition of Li+ leads to intense cationized drug species. Complex biological samples were cleaned up using a 96-well solid-phase extraction plate, and the purified samples were automatically spotted by a pipetting robot. To obtain a suitable data analysis procedure for the quantitative analysis of drugs by MALDI-TOF mass spectrometry, various data processing parameters were evaluated on our two model drugs lopinavir and ritonavir. Finally, and most importantly, it is shown that the above-described procedure can be successfully applied to quantify clinically relevant concentrations of lopinavir, an HIV protease inhibitor, in extracts of small numbers of peripheral blood mononuclear cells (1 x 10(6)).     

Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry

Glycopeptides prepared from 1 nmol of a mixture of glycoproteins, transferrin, and ribonuclease B by lysylendopeptidase digestion were isolated by lectin and cellulose column chromatographies, and then they were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry which enables the performance of MS ( n ) analysis. The lectin affinity preparation of glycopeptides with Sambucus nigra agglutinin and concanavalin A provides the glycan structure outlines for the sialyl linkage and the core structure of N-glycans. Such structural estimation was confirmed by MALDI-TOF MS and MALDI-QIT-TOF MS/MS. Amino acid sequences and location of glycosylation sites were determined by MALDI-QIT-TOF MS/MS/MS. Taken together, the combination of lectin column chromatography, MALDI-TOF MS, and MALDI-QIT-TOF MS ( n ) provides an easy way for the structural estimation of glycans and the rapid analysis of glycoproteomics.     

Approach for determining protein ubiquitination sites by MALDI-TOF mass spectrometry

Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the epsilon-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining posttraslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic peptides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves.     

Integrated microfluidic system enabling (bio)chemical reactions with on-line MALDI-TOF mass spectrometry

A continuous flow micro total analysis system (micro-TAS) consisting of an on-chip microfluidic device connected to a matrix assisted laser desorption ionization [MALDI] time-of-flight [TOF] mass spectrometer (MS) as an analytical screening system is presented. Reaction microchannels and inlet/outlet reservoirs were fabricated by powderblasting on glass wafers that were then bonded to silicon substrates. The novel lab-on-a-chip was realized by integrating the microdevice with a MALDI-TOFMS standard sample plate used as carrier to get the microfluidic device in the MALDI instrument. A novel pressure-driven pumping mechanism using the vacuum of the instrument as a driving force induces flow in the reaction microchannel in a self-activating way. Organic syntheses as well as biochemical reactions are carried out entirely inside the MALDI-MS ionization vacuum chamber and analyzed on-line by MALDI-TOFMS in real time. The effectiveness of the micro-TAS system has been successfully demonstrated with several examples of (bio)chemical reactions.     

Synthesis and characterization of novel triblock copolymers comprising poly(tetrahydrofuran) as a central block and poly(-benzyl -glutamate)s as outer blocks

BACKGROUND: Bioactive and biodegradable polyurethanes (BDPUs) have drawn much attention in recent years. As part of the research program to search for novel prepolymers for BDPUs, a study was carried out on the synthesis and characterization of triblock copolymers comprising poly(tetrahydrofuran) as a central block and poly(?-benzyl L-glutamate)s as outer blocks. RESULTS: A new macroinitiator terminated with phenylalanine was first prepared from the condensation of a distal hydroxy poly(tetrahydrofuran) with N-tert-butoxycarbonyl L-phenylalanine in the presence of dicyclohexylcarbodiimide, followed by removing the protecting group. Then, it was employed to initiate the ring-opening polymerization of ?-benzyl L-glutamate N-carboxyanhydride in varying feeding ratios to give rise to the targeted triblock copolymers. CONCLUSIONS: The length of the outer poly(?-benzyl L-glutamate) blocks was well tailored by varying the monomers to macroinitiator feeding ratio. All the triblock copolymers exhibited a nearly symmetrical and unimodal molecular weight distribution while only one distinct glass transition temperature was evidenced from -10°C to 25°C.     

Detection of plasmid insertion in Escherichia coli by MALDI-TOF mass spectrometry

A proteomics approach is reported for the rapid recognition of genetically modified Escherichia coli bacteria. The approach targets a class of proteins required for genetic manipulation of bacteria with plasmids and alleviates the need to construct extensive libraries of toxins and other predicted payload proteins. Detection was performed using MALDI-TOF MS to monitor peptide products after an on-probe enzymatic digestion. Digestion products were identified by searching their postsource decay spectra using MASCOT. A 5 min digestion time was required to observe peptide products from the genetic insert as well as the host bacterium. This proteomics approach enables rapid detection of genetic manipulation along with information about the host organism, both of which have forensic applications.