Ion trap-Fourier Transform Mass Spectrometer Research Articles

Increased cell proliferation and differential protein expression induced by low-level Er:YAG laser irradiation in human gingival fibroblasts: proteomic analysis.

Erbium-doped yttrium aluminum garnet (Er:YAG) laser treatment has demonstrated favorable wound healing effect after periodontal therapy. One of the reasons may be the positive biological effect of the low-level laser on the irradiated tissues, although the mechanism remains unclear. The aim of this study was to investigate the effect of low-level Er:YAG laser irradiation on cell proliferation and laser-induced differential expression of proteins in human gingival fibroblasts (HGFs) by proteomic analysis. In the first experiment, HGFs were exposed to low-level Er:YAG laser irradiation and the laser-induced cell proliferation and damage were evaluated on day 3. In the second experiment, proteomic analysis was performed on day 1 after irradiation. The peptides prepared from HGFs were analyzed by a hybrid ion trap-Fourier transform mass spectrometer, Mascot search engine, and UniProtKB database. A significant increase in cell proliferation without cell damage after irradiation was observed. Among the total identified 377 proteins, 59 proteins, including galectin-7, which was associated with the process of wound healing, were upregulated and 15 proteins were downregulated in laser-treated HGFs. In the third experiment, the increase in messenger RNA (mRNA) and protein expression of galectin-7 in the irradiated HGFs was validated by various analytical techniques. In addition, the effect of recombinant human galectin-7 on the modulation of HGFs proliferation was confirmed. The results indicate that low-level Er:YAG laser irradiation can promote HGF proliferation and induce a significant change in protein expression and the upregulation of galectin-7 expression may partly contribute to the increase in cell proliferation.

Protein Dynamics in Iron-Starved Mycobacterium tuberculosis Revealed by Turnover and Abundance Measurement Using Hybrid-Linear Ion Trap-Fourier Transform Mass Spectrometry

To study the proteome response of Mycobacterium tuberculosis H37Rv to a change in iron level, iron-starved late-log-phase cells were diluted in fresh low- and high-iron media containing [ (15)N]-labeled asparagine as the sole nitrogen source for labeling the proteins synthesized upon dilution. We determined the relative protein abundance and protein turnover in M. tuberculosis H37Rv under these two conditions. For measurements, we used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation. While relative protein abundance analysis shows that only 5 proteins were upregulated by high iron, 24 proteins had elevated protein turnover for the cells in the high-iron medium. This suggests that protein turnover is a sensitive parameter to assess the proteome dynamics. Cluster analysis was used to explore the interconnection of protein abundance and turnover, revealing coordination of the cellular processes of protein synthesis, degradation, and secretion that determine the abundance and allocation of a protein in the cytosol and the extracellular matrix of the cells. Further potential utility of the approach is discussed.

Determination of Global Protein Turnover in Stressed Mycobacterium Cells Using Hybrid-Linear Ion Trap-Fourier Transform Mass Spectrometry

We determined the global protein turnover profiles for Mycobacterium smegmatis under acid shock and iron starvation conditions using a simple (15)N isotope doping technique and a complete medium replacement method for chasing. We used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation to measure protein turnover values for 151 proteins over a dynamic range of 3 orders of magnitude ranging from about 0.2 to 500. Of these 151 proteins, 31 had significant protein turnover changes (p <0.05) at both stress conditions and had protein turnover values increased or decreased by more than 2-fold under at least one stress condition. Protein turnover increased under acid shock for 28 of the 31 proteins but decreased under iron starvation for all the 31 proteins. Only two proteins had protein turnover lowered by more than 2-fold (p <0.05) under both stress conditions, including an ATP synthase F1 beta subunit (MSMEG4921; AtpD) and a catalase/peroxidase (MSMEG6346; KatG). KatG is required for in vivo activation of isoniazid to be bacterialcidal. Decrease of KatG protein turnover under both stress conditions supports the view that isoniazid may induce a dormancy program in mycobacteria, which in turn limits the efficacy of this drug against dormant subpopulation of mycobacteria. Thus, measuring protein turnover in stressed Mycobacterium cells has implications in understanding drug action and resistance mechanisms.

Monitoring of Glycoprotein Products in Cell Culture Lysates Using Lectin Affinity Chromatography and Capillary HPLC Coupled to Electrospray Linear Ion Trap-Fourier Transform Mass Spectrometry (LTQ/FTMS)

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.

Analysis of the low molecular weight serum peptidome using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer

Advances in proteomics are continuing to expand the ability to analyze the serum proteome. In recent years, it has been realized that in addition to the circulating proteins, human serum also contains a large number of peptides. Many of these peptides are believed to be fragments of larger proteins that have been at least partially degraded by various enzymes such as metalloproteases. Identifying these peptides from a small amount of serum/plasma is difficult due to the complexity of the sample, the low levels of these peptides, and the difficulties in getting a protein identification from a single peptide. In this study, we modified previously published protocols for using centrifugal ultrafiltration, and unlike past studies did not digest the filtrate with trypsin with the intent of identifying endogenous peptides with this method. The filtrate fraction was concentrated and analyzed by a reversed phase-high performance liquid chromatography system connected to a nanospray ionization hybrid ion trap-Fourier transform mass spectrometer (LTQ-FTMS). The mass accuracy of this instrument allows confidence for identifying the protein precursors by a single peptide. The utility of this approach was demonstrated by the identification of over 300 unique peptides with 2 ppm or better mass accuracy per serum sample. With confident identifications, the origin and function of native serum peptides can be more seriously explored. Interestingly, over 34 peptide ladders were observed from over 17 serum proteins. This indicates that a cascade of proteolytic processes affects the serum peptidome. To examine whether this result was an artifact of serum, matched plasma and serum samples were analyzed with similar peptide ladders found in each.

Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system

A mass spectrometry analysis of the yeast proteome shows that complex mixture analysis is not limited by sensitivity but by a combination of dynamic range and by effective sequencing speed.

Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications

Chromatin is regulated at many different levels, from higher-order packing to individual nucleosome placement. Recent studies have shown that individual histone modifications, and combinations thereof, play a key role in modulating chromatin structure and gene activity. Reported here is an analysis of Arabidopsis histone H3 modifications by nanoflow-HPLC coupled to electrospray ionization on a hybrid linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS). We find that the sites of acetylation and methylation, in general, correlate well with other plants and animals. Two well-studied modifications, dimethylation of Lys-9 (correlated with silencing) and acetylation of Lys-14 (correlated with active chromatin) while abundant by themselves were rarely found on the same histone H3 tail. In contrast, dimethylation at Lys-27 and monomethylation at Lys-36 were commonly found together. Interestingly, acetylation at Lys-9 was found only in a low percentage of histones while acetylation of Lys-14 was very abundant. The two histone H3 variants, H3.1 and H3.2, also differ in the abundance of silencing and activating marks confirming other studies showing that the replication-independent histone H3 is enriched in active chromatin.

Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation.

MS-based proteomics usually involves the fragmentation of tryptic peptides (tandem MS or MS(2)) and their identification by searching protein sequence databases. In ion trap instruments fragments can be further fragmented and analyzed, a process termed MS/MS/MS or MS(3). Here, we report that efficient ion capture in a linear ion trap leads to MS(3) acquisition times and spectra quality similar to those for MS(2) experiments with conventional 3D ion traps. Fragmentation of N- or C-terminal ions resulted in informative and low-background spectra, even at subfemtomol levels of peptide. Typically C-terminal ions are chosen for further fragmentation, and the MS(3) spectrum greatly constrains the C-terminal amino acids of the peptide sequence. MS(3) spectra allow resolution of ambiguities in identification, a crucial problem in proteomics. Because of the sensitivity and rapid scan rates of the linear ion trap, several MS(3) spectra per peptide can be obtained even when sequencing very complex mixtures. We calculate the probability that an experimental MS(3) spectrum originates from fragmentation of a given N- or C-terminal ion of a peptide under consideration. This MS(3) identification score can be combined with the MS(2) scores of the precursor peptide from existing search engines. When MS(3) is performed on the linear ion trap-Fourier transform mass spectrometer combination, accurate peptide masses further increase confidence in peptide identification.