Talat Yalcin

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.

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 run 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 ∼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.

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 solvents 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.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for the Analysis of Polydienes

An analytical method based on matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry has been developed to provide information on oligomer structure, average molecular weight, and molecular weight distributions of polydienes (e.g., polybutadiene and polyisoprene), an important class of industrial polymers. This MALDI method involves the use of all-trans-retinoic acid as the matrix, copper (II) nitrate as the cationization reagent, and tetrahydrofuran as the solvent. The incorporation of this copper salt generates Cu+ adducts with the polymer chains. It also improves the signal strength and extends the upper mass range when used with all-trans-retinoic acid, as compared to silver nitrate. With this formulation, it is demonstrated that polybutadienes of narrow polydispersity with masses up to 300,000 u and polyisoprenes of narrow polydispersity with masses up to 150,000 u can be analyzed. The upper molecular weight limit is set by the requirement of using higher matrix-to-polymer ratios with increasing polymer molecular weight, to the point where the instrument can no longer detect the small quantity of polymer present in the matrix host. It is also shown that this sample preparation generates previously unreported adduction behavior. The practical implications of this adduction behavior on polymer structural analysis, accuracy of molecular weight determination, and the upper molecular weight limit of oligomer resolution are discussed. It is illustrated that, in a linear time-lag focusing MALDI instrument, oligomer resolution can be obtained for polydienes with molecular weights up to 24,000, providing structural confirmation of the end-groups and the repeat unit. The average molecular weights of a number of polydienes of narrow polydispersity determined by MALDI are compared to those obtained by gel permeation chromatography, and discrepancies are noted.

A Systematic Study of Acidic Peptides for b-Type Sequence Scrambling

A systematic study was carried out to examine the effects of acidic amino acid residues and the position of the acidic group on the cyclization of b ions. The study utilized the model C-terminal amidated peptides XAAAAAA, AXAAAAA, AAXAAAA, AAAXAAA, AAAAXAA, AAAAAXA, AAAAAAX, XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX, where X is a glutamic acid (E) or aspartic acid (D) residue. The CID mass spectra of bn (where n = 7 and 8) ions derived from XAAAAAA, AAAXAAA, AAAAAAX and XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX exhibited very similar fragmentation patterns for both the glutamic and the aspartic acid peptide series. The CID mass spectra of MH+ derived from model peptides presented substantial direct and non-direct sequence b ions. The results indicate that b ions produced from acidic peptides can also undergo head-to-tail cyclization, which is the reason for the formation of the non-direct sequence b ions. The b ion spectra derived from the peptides became more complex as the number of acidic residues in the peptides increased. Side chains of glutamic and aspartic acid did not inhibit the cyclization of the b ions. Substantial water elimination was observed in all CID spectra of b7 and b8 ions. Finally, the preferential cleavage of glutamic or aspartic acid residues from macrocyclic structures of b ions was also investigated under various collision energy conditions.

Analysis of polymers using evolved-gas and direct-pyrolysis techniques

Thermal analysis of polystyrene, poly(p-methylstyrene) and poly(α-methylstyrene) has been carried out using evolved-gas analysis by infrared and mass spectrometry, and direct-pyrolysis analysis by mass spectrometric techniques. Evolved-gas analysis, both by infrared and mass spectrometry, reveals features due mainly to the corresponding monomers or stable, volatile, and low relative molecular mass degradation products. In direct-pyrolysis mass spectrometry, however, primary decomposition products and heavier fragments such as dimers and trimers can also be detected. The ion-temperature profiles of the corresponding monomer ions reveal information about the thermal stability of the polymers.

Genome-wide identification of genes that play a role in boron stress response in yeast

Boron is an essential micronutrient for plants and it is either necessary or beneficial for animals. Studies identified only few genes related to boron metabolism thus far and details of how boron is imported into cells and used in cell metabolism are largely unknown. In order to identify genes that play roles in boron metabolism, we screened the entire set of yeast haploid deletion mutants and identified 6 mutants that were resistant to toxic levels of boron, and 21 mutants that were highly sensitive to boron treatment. Furthermore, we performed a proteomic approach to identify additional proteins that are significantly up-regulated by boron treatment. Our results revealed many genes and pathways related to boron stress response and suggest a possible link between boron toxicity and translational control.

Dissociation of protonated phenylthiohydantoin‐amino acids and phenylthiocarbamoyl‐dipeptides

The N-terminal phenylthiocarbamoyl (PTC) derivatives of peptides and the phenylthiohydantoin (PTH) derivatives of amino acids are the two major types of products generated in the Edman protein sequencing method. Understanding the fragmentation pathways of these species should facilitate structural elucidation and chemical identification based on the fragment ion mass spectra, particularly when mass spectrometry is combined with the Edman sequencer for the analysis of non-standard and modified amino acids. In this study, dissociation of the protonated PTH-X (where X=Thr, Ser, Trp and Tyr), PTH-Gly, PTC-X-Leu and PTC-Gly-Leu in electrospray ionization mass spectrometry was examined to investigate whether there is any isomerization of PTC to PTH derivatives in the gas phase during the fragmentation. It is shown that dissociation of the protonated PTH-X proceeds via hydrogen transfer from the side-chain of the amino acid to the PTH moiety with the elimination of the side-chain as a neutral species. The ions at m/z 193 formed from the source fragmentation of the protonated PTH-X are found to have the same structure and fragmentation pathways. The presence of this m/z 193 ion and its collisionally induced dissociation (CID) spectrum are unique for the PTH derivatives and they can be used to detect the presence of the PTH ions. It is shown that there is no isomerization of the thiazolone ions to the PTH ions during the dissociation of PTC-X-Leu (in this case, the b1 ions from PTC-X-Leu are believed to have the protonated thiazolone structure). In addition, comparative studies of CID spectra of PTH-X and PTH-Gly or PTC-X-Leu and PTC-Gly-Leu are presented. The proposed fragmentation mechanisms for the protonated PTH and PTC derivatives and the m/z 193 ions are given.

Non-Direct Sequence Ions in the Tandem Mass Spectrometry of Protonated Peptide Amides—an Energy-Resolved Study

AbstractThe fragmentation reactions of the MH+ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of bn ions, including significant loss of NH3 from the MH+ ions. Further fragmentation of these bn ions occurs following macrocyclization/ring opening leading in many cases to bn ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies. Figureᅟ

Protonated Dipeptide Losses from b 5 and b 4 Ions of Side Chain Hydroxyl Group Containing Pentapeptides

AbstractIn this study, C-terminal protonated dipeptide eliminations were reported for both b5 and b4 ions of side chain hydroxyl group (–OH) containing pentapeptides. The study utilized the model C-terminal amidated pentapeptides having sequences of XGGFL and AXVYI, where X represents serine (S), threonine (T), glutamic acid (E), aspartic acid (D), or tyrosine (Y) residue. Upon low-energy collision-induced dissociation (CID) of XGGFL (where X = S, T, E, D, and Y) model peptide series, the ions at m/z 279 and 223 were observed as common fragments in all b5 and b4 ion (except b4 ion of YGGFL) mass spectra, respectively. By contrast, peptides, namely SMeGGFL-NH2 and EOMeGGFL-NH2, did not show either the ion at m/z 279 or the ion at m/z 223. It is shown that the side chain hydroxyl group is required for the possible mechanism to take place that furnishes the protonated dipeptide loss from b5 and b4 ions. In addition, the ions at m/z 295 and 281 were detected as common fragments in all b5 and b4 ion (except b4 ion of AYVYI) mass spectra, respectively, for AXVYI model peptide series. The MS4 experiments exhibited that the fragment ions at m/z 279, 223, 295, and 281 entirely reflect the same fragmentation behavior of [M + H]+ ion generated from commercial dipeptides FL-OH, GF-OH, YI-OH, and VY-OH. These novel eliminations reported here for b5 and b4 ions can be useful in assigning the correct and reliable peptide sequences for high-throughput proteomic studies. Figureᅟ