Single-chip detector for electron spin resonance spectroscopy.

We have realized an innovative integrated detector for electron spin resonance spectroscopy. The microsystem, consisting of an LC oscillator, a mixer, and a frequency division module, is integrated onto a single silicon chip using a conventional complementary metal-oxide-semiconductor technology. The implemented detection method is based on the measurement of the variation of the frequency of the integrated LC oscillator as a function of the applied static magnetic field, caused by the presence of a resonating sample placed over the inductor of the LC-tank circuit. The achieved room temperature spin sensitivity is about 10(10) spinsGHz(12) with a sensitive volume of about (100 microm)(3).

Assessment of the molecular weight distribution of tannin fractions through MALDI-TOF MS analysis of protein-tannin complexes.

An innovative mass spectrometry method was developed for determining mass distributions of tannin fractions that cannot be approached through direct MALDI-TOF analysis. It was applied to three procyanidin fractions with average degrees of polymerizations = 3, 9, and 28, respectively, and one gallotannin fraction (Tara tannin). The proposed approach consists of MALDI-TOF analysis of the soluble complexes formed between these tannin fractions and bovine serum albumin (BSA). Complexes were detected as an unresolved "hump" following the BSA signal, and spectra were mathematically processed to determine the parameters relative to the protein-tannin complexes, which are the number-average molecular weight (Mn), the weight-average molecular weight (Mw), and the polydispersity index (PI) for each tannin fraction. Regarding condensed tannins, results are consistent with those of the standard method (thiolysis followed by HPLC separation) for all tested fractions. The method was successfully applied to a hydrolyzable tannin fraction but no standard method is available for comparison.

Laser desorption ionization and MALDI time-of-flight mass spectrometry for low molecular mass polyethylene analysis.

Polyethylene's inert nature and difficulty to dissolve in conventional solvents at room temperature present special problems for sample preparation and ionization in mass spectrometric analysis. We present a study of ionization behavior of several polyethylene samples with molecular masses up to 4000 Da in laser desorption ionization (LDI) time-of-flight mass spectrometers equipped with a 337 nm laser beam. We demonstrate unequivocally that silver or copper ion attachment to saturated polyethylene can occur in the gas phase during the UV LDI process. In LDI spectra of polyethylene with molecular masses above approximately 1000 Da, low mass ions corresponding to metal-alkene structures are observed in addition to the principal distribution. By interrogating a well-characterized polyethylene sample and a long chain alkane, C94H190, these low mass ions are determined to be the fragmentation products of the intact metal-polyethylene adduct ions. It is further illustrated that fragmentation can be reduced by adding matrix molecules to the sample preparation.

Structural analysis of polymer end groups by electrospray ionization high-energy collision-induced dissociation tandem mass spectrometry

Chemical structures of polymer end groups play an important role in determining the functional properties of a polymeric system. We present a mass spectrometric method for determining end group structures. Polymeric ions are produced by electrospray ionization (ESI), and they are subject to source fragmentation in the ESI interface region to produce low-mass fragment ions. A series of source-fragment ions containing various numbers of monomer units are selected for high-energy collision-induced dissociation (CID) in a sector/time-of-flight tandem mass spectrometer. It is shown that high-energy CID spectra of source-induced fragment ions are very informative for end group structure characterization. By comparing the CID spectra of fragment ions with those of known chemicals, it is possible to unambiguously identify the end group structures. The utility of this technique is illustrated for the analysis of two poly(ethylene glycol)-based slow-releasing drugs where detailed structural characterization is of significance for drug formulation, quality control, and regulatory approval. Practical issues related to the application of this method are discussed.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer analysis: solvent effect in sample preparation.

The success of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for the characterization of polymer structures and for the determination of average molecular weights and distributions depends on the use of a proper sample/matrix preparation protocol. This work examines the effect of solvents, particularly solvent mixtures, used to prepare polymer, matrix, and cationization reagent solutions, on MALDI analysis. It is shown that the use of solvent mixtures consisting of polymer solvent does not have a significant effect on the molecular weight determination of polystyrene 7000 and poly(methyl methacrylate) 3750. However, solvent mixtures containing a polymer nonsolvent can affect the signal reproducibility and cause errors in average weight measurement. This solvent effect was further investigated by using confocal laser fluorescence microscopy in conjunction with the use of a fluorescein-labeled polystyrene. It is demonstrated that sample morphology and polymer distribution on the probe can be greatly influenced by the type of solvents used. For sample preparation in MALDI analysis of polymers, it is important to select a solvent system that will allow matrix crystallization to take place prior to polymer precipitation. The use of an excess amount of any polymer nonsolvent should be avoided.

MALDI mass spectrometry combined with avidin-biotin chemistry for analysis of protein modifications.

A general mass spectrometric method that combines purification and analysis in one step is described for the rapid and sensitive determination of protein modification that involves covalent attachment of a modifying group. In this method, the modifying group is first labeled with a biotin moiety, and the covalent interaction of this group with the targeted protein results in a biotinylated product. The modified protein can then be subjected to enzymatic digestion, followed by the isolation of the biotinylated peptide based on a previously described MALDI method incorporating the avidin-biotin interaction (Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 3382-3387). To illustrate the validity of the method, a study of a model system was undertaken, involving the interaction between avian skeletal muscle troponin C and a sulfhydryl-specific biotinylation reagent. It is shown that isolation of a modified peptide with an immobilized avidin product could be achieved, even in the presence of an excess of contaminating protein. Exoproteases could be added to the crude tryptic digest to generate peptide ladders, each containing biotin, which could be analyzed by the avidin-biotin/MALDI method for sequence information. Complementary sequence information could be obtained from the application of this technique in a tandem sector/time-of-flight mass spectrometer for MALDI MS/MS analysis, which allowed for the identification of the modification site.

Analysis of the accuracy of determining average molecular weights of narrow polydispersity polymers by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) can be used to determine number- and weight-average molecular weights of narrow polydispersity polymers. In this work, several possible sources of error in determining molecular weights of polymers with narrow polydispersity by MALDI-TOFMS are rigorously examined. These include the change in polymer distribution function, broadening or narrowing of the overall distribution, and the truncation of selected oligomer peaks within a distribution (i.e., the oligomer peaks at the high- and low-mass tails expected to be observed are not detected). These variations could be brought about by a limited detection sensitivity, background interference, and/or mass discrimination of oligomer analysis in MALDI-TOFMS. For narrow polydispersity polystyrenes, it is shown that by using an appropriate MALDI matrix and sample preparation protocol and a sensitive ion detection instrument, no systematic errors from these possible variations were detected within the experimental precision (0.5% relative standard deviation) of the MALDI method. It is concluded that MALDI mass spectrometry can provide accurate molecular weight and molecular weight distribution information for narrow polydispersity polymers, at least for polystyrenes examined in this work. The implications of this finding for polymer analysis are discussed.

Ion chemistry of protonated lysine derivatives.

Protonated lysine fragments primarily by elimination of the epsilon-amino group as ammonia to form an ion of m/z 130 and to a minor extent by elimination of H2O to form an ion of m/z 129. Protonated lysine derivatives such as lysine beta-naphthylamide and H-Lys-Gly-OH show more pronounced formation of m/z 129 while protonated derivatives such as N alpha-Ac-Lys-X (X = OH, OMe, NHMe) and H-Gly-Lys-X (X = OH, NHCH2COOH) also show formation of m/z 129 in both metastable ion and collision-induced fragmentation. In both the latter systems m/z 129 is formed by sequential loss of HX followed by loss of ketene for the N-acetyl derivatives or the glycine residue for the N-glycyl derivatives. Although the m/z 129 ion is nominally an acylium ion, its metastable ion characteristics and collision-induced dissociation mass spectrum are very similar to those of protonated alpha-amino-epsilon-caprolactam. It is concluded that this lactam is formed from the lysine derivatives by interaction of the amino group of the lysine side-chain with the lysine carbonyl function as HX departs. Protonated N alpha-methyllysine and N alpha-dimethyllysine fragment exclusively by elimination of CH3NH2 and (CH3)2NH, respectively. Evidence is presented that the stable structure of the m/z 130 ion so formed is protonated pipecolic acid. Both the protonated alpha-amino-epsilon-caprolactam and protonated pipecolic acid ions fragment further primarily to [C5H10N]+ (m/z 84), a low mass ion commonly observed in the spectra of lysine-containing peptides.

The structure and fragmentation of B n (n≥3) ions in peptide spectra.

The unimolecular and low energy collision-induced fragmentation reactions of the MH(+) ions of N-acetyl-tri-alanine, N-acetyl-tri-alanine methyl ester, N-acetyl-tetra-alanine, tetra-alanine, penta-alanine, hexa-glycine, and Leu-enkephalin have been studied with a particular emphasis on the formation and fragmentation of B n (n=3,4,5) ions. In addition, the metastable ion fragmentation reactions of protonated tetra-glycine, penta-glycine, and Leu-enkephalin amide have been studied. B n ions are prominent stable species in all spectra. The B n ions fragment, in part, by elimination of CO to form A n ions; this reaction occurs on the metastable ion time scale with a substantial release of kinetic energy (T 1/2=0. 3-0. 5 eV) that indicates that a stable configuration of the B n ion fragments by way of a reacting configuration that is higher in energy than the fragmentation products, A n + CO. Ab initio calculations strongly suggest that the stable configuration of the B3 and B4 ions is a protonated oxazolone formed by interaction of the developing charge with the next-nearest carbonyl group as HX is lost from the protonated species H-(Yyy) n -X · H(+). The higher B n ions also fragment, in part, to form the next-lower B ion, presumably in its stable protonated oxazolone form. This reaction is rationalized in terms of the three-dimensional structure of the B n ions and it is proposed that the neutral eliminated is an α-lactam.

Why Are B ions stable species in peptide spectra?

Protonated amino acids and derivatives RCH(NH2)C(+O)X · H(+) (X = OH, NH2, OCH3) do not form stable acylium ions on loss of HX, but rather the acylium ion eliminates CO to form the immonium ion RCH = NH 2 (+) . By contrast, protonated dipeptide derivatives H2NCH(R)C(+O)NHCH(R')C(+O)X · H(+) [X = OH, OCH3, NH2, NHCH(R″)COOH] form stable B2 ions by elimination of HX. These B2 ions fragment on the metastable ion time scale by elimination of CO with substantial kinetic energy release (T 1/2 = 0.3-0.5 eV). Similarly, protonated N-acetyl amino acid derivatives CH3C(+O)NHCH(R')C(+O)X · H(+) [X = OH, OCH3, NH2, NHCH(R″)COOH] form stable B ions by loss of HX. These B ions also fragment unimolecularly by loss of CO with T 1/2 values of ∼ 0.5 eV. These large kinetic energy releases indicate that a stable configuration of the B ions fragments by way of activation to a reacting configuration that is higher in energy than the products, and some of the fragmentation exothermicity of the final step is partitioned into kinetic energy of the separating fragments. We conclude that the stable configuration is a protonated oxazolone, which is formed by interaction of the developing charge (as HX is lost) with the N-terminus carbonyl group and that the reacting configuration is the acyclic acylium ion. This conclusion is supported by the similar fragmentation behavior of protonated 2-phenyl-5-oxazolone and the B ion derived by loss of H-Gly-OH from protonated C6H5C(+O)-Gly-Gly-OH. In addition, ab initio calculations on the simplest B ion, nominally HC(+O)NHCH2CO(+), show that the lowest energy structure is the protonated oxazolone. The acyclic acylium isomer is 1.49 eV higher in energy than the protonated oxazolone and 0.88 eV higher in energy than the fragmentation products, HC(+O)N(+)H = CH2 + CO, which is consistent with the kinetic energy releases measured.