MS and MS/MS spectra of glycopeptides from 10 pmol gel-separated human transferrin.

<p>(A) Mass spectrum of the glycopeptides of human transferrin. (B) The enlarged view of Figure 2A, which shows low-abundance glycopeptides more clearly. (C) Mass spectrum of the parent ion of m/z 3684.6 obtained in TOF/TOF mode. The difference between 3682.3, 3391.6 and 3100.5 is about 291. (D) MS/MS spectrum from the precursor indicated by arrow in Figure 2C. The mass of [M_pep+H]⁺ was 1476.7. Glycan structures were deduced based on the difference between adjacent signals and its biosynthesis process.</p

2-DE image of 10 µL human serum and mass spectra of glycopeptides of 2-DE separated α1-antitrypsin.

<p>(A) 2-DE image of 10 µL human serum. Two largest spots of α1-antitrypsin were chosen for glycosylation analysis and named as A1PI-1 and A1PI-2. (B)–(C): Mass spectra of the glycopeptides of A1PI-1 and A1PI-2 in the mass range of 5500-7000 Dalton. Data were shown in average value. Compared with A1PI-1, the relative abundance of diantennary N-glycans indicated by arrows in A1PI-2 increased significantly, while the triantennary N-glycans decreased significantly.</p

Relative abundance (%) of the glycans identified at Asn107 of human α1-antitrypsin ^a.

a<p>Approximate abundance was estimated using MS signal intensities from a single analysis.</p

MS and MS/MS spectra of deglycosylated peptides and desialylated glycans of human transferrin.

<p>(A) Mass spectrum of deglycosylated peptides of human transferrin, m/z 1477.681, 2516.072 and 3530.644 were identified as deglycosylated peptides. (B) MS/MS spectrum of m/z 2516 indicated by arrow in Figure 3A. (C) Mass spectrum of desialylated glycans of human transferrin. The MS signals were [M+Na]⁺ ions in average values, and corresponding structures were also shown.</p

The mass of potential glycopeptides of human transferrin calculated from that of deglycosylated peptides and desialylated glycans.

<p>M_PG: Mass of potential glycopeptides.</p><p>Note: The measured masses of the glycopeptides of human transferrin are 3393.0, 3684.6, 4049.4, 4142.9, 4432.1, 4576.6, 4724.0, 4871.2, 5088.7, 5234.1, 5381.8, 5525.9, 5738.8 and 5884.1. The values in bold are identified glycopeptides.</p

Correction of Errors in Tandem Mass Spectrum Extraction Enhances Phosphopeptide Identification

The tandem mass spectrum extraction of phosphopeptides is more difficult and error-prone than that of unmodified peptides due to their lower abundance, lower ionization efficiency, the cofragmentation with other high-abundance peptides, and the use of MS³ on MS² fragments with neutral losses. However, there are still no established methods to evaluate its correctness. Here we propose to identify and correct these errors via the combinatorial use of multiple spectrum extraction tools. We evaluated five free and two commercial extraction tools using Mascot and phosphoproteomics raw data from LTQ FT Ultra, in which RawXtract 1.9.9.2 identified the highest number of unique phosphopeptides (peptide expectation value <0.05). Surprisingly, ProteoWizzard (v. 3.0.3476) extracted wrong precursor mass for most MS³ spectra. Comparison of the top three free extraction tools showed that only 54% of the identified spectra were identified consistently from all three tools, indicating that some errors might happen during spectrum extraction. Manual check of 258 spectra not identified from all three tools revealed 405 errors of spectrum extraction with 7.4% in selecting wrong precursor charge, 50.6% in selecting wrong precursor mass, and 42.1% in exporting MS/MS fragments. We then corrected the errors by selecting the best extracted MGF file for each spectrum among the three tools for another database search. With the errors corrected, it results in the 22.4 and 12.2% increase in spectrum matches and unique peptide identification, respectively, compared with the best single method. Correction of errors in spectrum extraction improves both the sensitivity and confidence of phosphopeptide identification. Data analysis on nonphosphopeptide spectra indicates that this strategy applies to unmodified peptides as well. The identification of errors in spectrum extraction will promote the improvement of spectrum extraction tools in future

Correction of Errors in Tandem Mass Spectrum Extraction Enhances Phosphopeptide Identification

The tandem mass spectrum extraction of phosphopeptides is more difficult and error-prone than that of unmodified peptides due to their lower abundance, lower ionization efficiency, the cofragmentation with other high-abundance peptides, and the use of MS³ on MS² fragments with neutral losses. However, there are still no established methods to evaluate its correctness. Here we propose to identify and correct these errors via the combinatorial use of multiple spectrum extraction tools. We evaluated five free and two commercial extraction tools using Mascot and phosphoproteomics raw data from LTQ FT Ultra, in which RawXtract 1.9.9.2 identified the highest number of unique phosphopeptides (peptide expectation value <0.05). Surprisingly, ProteoWizzard (v. 3.0.3476) extracted wrong precursor mass for most MS³ spectra. Comparison of the top three free extraction tools showed that only 54% of the identified spectra were identified consistently from all three tools, indicating that some errors might happen during spectrum extraction. Manual check of 258 spectra not identified from all three tools revealed 405 errors of spectrum extraction with 7.4% in selecting wrong precursor charge, 50.6% in selecting wrong precursor mass, and 42.1% in exporting MS/MS fragments. We then corrected the errors by selecting the best extracted MGF file for each spectrum among the three tools for another database search. With the errors corrected, it results in the 22.4 and 12.2% increase in spectrum matches and unique peptide identification, respectively, compared with the best single method. Correction of errors in spectrum extraction improves both the sensitivity and confidence of phosphopeptide identification. Data analysis on nonphosphopeptide spectra indicates that this strategy applies to unmodified peptides as well. The identification of errors in spectrum extraction will promote the improvement of spectrum extraction tools in future

Evaluation of the Effect of Trypsin Digestion Buffers on Artificial Deamidation

Nonenzymatic deamidation occurs readily under the condition of trypsin digestion, resulting in the identification of many artificial deamidation sites. To evaluate the effect of trypsin digestion buffers on artificial deamidation, we compared the three commonly used buffers Tris-HCl (pH 8), ammonium bicarbonate (ABC), and triethylammonium bicarbonate (TEAB), and ammonium acetate (pH 6), which was reported to reduce Asn deamidation. iTRAQ quantification on rat kidney tissue digested in these four buffers indicates that artificial Asn deamidation is produced in the order of ammonium acetate < Tris-HCl < ABC < TEAB, and Gln deamidation has no significant differences in all tested buffers. Label-free experiments show the same trend, while protein and unique peptide identification are comparable using these four buffers. To explain the differences of these four buffers in producing artificial Asn deamidation, we determined the half-life of Asn deamidation in these buffers using synthetic peptides containing -Asn-Gly- sequences. It is 51.4 ± 6.0 days in 50 mM of ammonium acetate (pH 6) at 37 °C, which is about 23, 104, and 137 times that in Tris-HCl, ABC, and TEAB buffers, respectively. In conclusion, ammonium acetate (pH 6) is more suitable than other tested buffers for characterizing endogenous deamidation and N-glycosylation

Irradiation of Epithelial Carcinoma Cells Upregulates Calcium-Binding Proteins That Promote Survival under Hypoxic Conditions

Hypoxia is thought to promote tumor radio-resistance via effects on gene expression in cancer cells that modulate their metabolism, proliferation, and DNA repair pathways to enhance survival. Here we demonstrate for the first time that under hypoxic condition A431 epithelial carcinoma cells exhibit increased viability when exposed to low-dose γ-irradiation, indicating that radiotherapy can promote tumor cell survival when oxygen supply is limited. When assessed using iTRAQ quantitative proteomics and Western blotting, irradiated tumor cells were observed to significantly up-regulate the expression of calcium-binding proteins CALM1, CALU, and RCN1, suggesting important roles for these mediators in promoting tumor cell survival during hypoxia. Accordingly, shRNA-knockdown of CALM1, CALU, and RCN1 expression reduced hypoxic tumor cell resistance to low-dose radiation and increased apoptosis. These data indicate that γ-irradiation of hypoxic tumor cells induces up-regulation of calcium-binding proteins that promote cancer cell survival and may limit the efficacy of radiotherapy in the clinic

Small Molecule Probe Suitable for <i>In Situ</i> Profiling and Inhibition of Protein Disulfide Isomerase

Proper folding of cellular proteins is assisted by protein disulfide isomerases (PDIs) in the endoplasmic reticulum of mammalian cells. Of the at least 21 PDI family members known in humans, the 57-kDa PDI has been found to be a potential therapeutic target for a variety of human diseases including cancer and neurodegenerative diseases. Consequently, small molecule PDI-targeting inhibitors have been actively pursued in recent years, and thus far, compounds possessing moderate inhibitory activities (IC₅₀ between 0.1 and 100 μM against recombinant PDI) have been discovered. In this article, by using <i>in situ</i> proteome profiling experiments in combination with <i>in vitro</i> PDI enzymatic inhibition assays, we have discovered a phenyl vinyl sulfonate-containing small molecule (<b>P1</b>; shown) as a relatively potent and specific inhibitor of endogenous human PDI in several mammalian cancer cells (e.g., GI₅₀ ∼ 4 μM). It also possesses an IC₅₀ value of 1.7 ± 0.4 μM in an <i>in vitro</i> insulin aggregation assay. Our results indicate <b>P1</b> is indeed a novel, cell-permeable small molecule PDI inhibitor, and the electrophilic vinyl sulfonate scaffold might serve as a starting point for future development of next-generation PDI inhibitors and probes