Identification of protein carbonylation sites by two-dimensional liquid chromatography in combination with MALDI- and ESI-MS

Oxidative stress is defined as excessive production of reactive oxygen species (ROS) overwhelming the cellular antioxidant defense systems and thereby damaging most constituents of cells including proteins. Reactive carbonyls, i.e. aldehydes, ketones and lactams, are a major class of irreversible oxidative protein modifications that are widely used as biomarkers of oxidative stress, aging and age-related diseases. Whereas carbonylated proteins can be studied by western blotting and ELISA, their site specific mapping still remains a challenging task due to their low abundance and insufficient ionization. Here, we present a new strategy to identify carbonylation sites in a bottom-up approach. Protein digests were derivatized with 2,4-dinitrophenyl hydrazine (DNPH) and separated by hydrophilic interaction chromatography (HILIC). Peptide-containing fractions were then analyzed by laser-desorption/ionization with DNPH as the reactive matrix, which favors DNP-labeled peptides. The mass list generated for each HILIC fraction, representing mostly DNP-modified peptides, was used in the subsequent nano reversed-phase chromatography (RPC) coupled on-line to an electrospray ionization Orbitrap mass spectrometer recording the tandem mass spectra in data dependent acquisition mode. This comprehensive two-dimensional HILIC×RPC-strategy was exemplified for tryptic digests of native bovine serum albumin (BSA) and β-lactoglobulin (β-LG), as well as their in vitro oxidized versions, i.e. oxBSA and oxβ-LG. In total, three carbonylation sites were identified in native β-LG, nine in native BSA, eleven in oxβ-LG and 32 in oxBSA.     

Identification of carbonylated peptides by tandem mass spectrometry using a precursor ion-like scan in negative ion mode

Reactive oxygen species (ROS) can oxidize proteins at almost any amino acid residue. Whereas some modifications are reversible within the cells, the higher oxidation states are especially irreversible. These irreversible post translational modifications are widely used as biomarkers of oxidative stress, such as protein carbonylation, which refers to aldehydes, ketones and lactams as 'reactive carbonyl groups'. This study relied on a set of synthetic peptides containing a C-terminal aldehyde (arginal) or modification with pyruvic acid (ketone) or 4-hydroxynonenal (aldehyde) at lysine or histidine residues, as well as peptides containing pyroglutamic acid (oxidation product of proline) and 2-amino-3-butyric acid (oxidation product of threonine). The carbonylation sites were specifically derivatized with 2,4-dinitrophenylhydrazine (DNPH) and the fragmentation behavior of the products investigated in electrospray ionization (ESI-) MS. Importantly, the DNPH-labeled carbonylated peptides showed favorable ionization behaviors in negative ion mode ESI, providing a sensitive detection method. Regular peptides were mostly discriminated under these conditions. Among the fragmentation techniques tested for the negatively charged ions, pulsed Q dissociation provided three diagnostic ions at m/z values 152.0, 163.1 and 179.0, specific for DNPH-modified peptides. These marker ions were successfully applied to detect the carbonylated model peptides in a spiked tryptic digest of bovine serum albumin and a complex protein mixture obtained from HeLa cells.     

Poplar under drought: comparison of leaf and cambial proteomic responses

The forest ecosystem is of particular importance from an economic and ecological perspective. However, the stress physiology of trees, perennial and woody plants, is far from being fully understood. For that purpose, poplar plants were exposed to drought; the plants exhibited commonly reported drought stress traits in the different plant tissues. Leafy rooted cuttings of poplar were investigated through a proteomic approach in order to compare the water constraint response of two plant tissues, namely leaf and cambium. Sampling was realized during the drought period at 2 time points with increased drought intensity and 7 days after rewatering. Our data show that there is a difference in the moment of response to the water constraint between the two tissues, cambium being affected later than leaves. In leaves, drought induced a decrease in rubisco content, and an increase in the abundance of light harvesting complex proteins as well as changes in membrane-related proteins. In the cambial tissue, the salient proteome pattern change was the decrease of multiple proteins identified as bark storage proteins. After rewatering, almost all changes in cambial proteome disappeared whereas a significant number of leaf proteins appeared to be differentially regulated only during the recovery from drought.     

Familial adenomatous polyposis-associated desmoids display significantly more genetic changes than sporadic desmoids

Desmoid tumours (also called deep or aggressive fibromatoses) are potentially life-threatening fibromatous lesions. Hereditary desmoid tumours arise in individuals affected by either familial adenomatous polyposis (FAP) or hereditary desmoid disease (HDD) carrying germline mutations in APC. Most sporadic desmoids carry somatic mutations in CTNNB1. Previous studies identified losses on 5q and 6q, and gains on 8q and 20q as recurrent genetic changes in desmoids. However, virtually all genetic changes were derived from sporadic tumours. To investigate the somatic alterations in FAP-associated desmoids and to compare them with changes occurring in sporadic tumours, we analysed 17 FAP-associated and 38 sporadic desmoids by array comparative genomic hybridisation and multiple ligation-dependent probe amplification. Overall, the desmoids displayed only a limited number of genetic changes, occurring in 44% of cases. Recurrent gains at 8q (7%) and 20q (5%) were almost exclusively found in sporadic tumours. Recurrent losses were observed for a 700 kb region at 5q22.2, comprising the APC gene (11%), a 2 Mb region at 6p21.2-p21.1 (15%), and a relatively large region at 6q15-q23.3 (20%). The FAP-associated desmoids displayed a significantly higher frequency of copy number abnormalities (59%) than the sporadic tumours (37%). As predicted by the APC germline mutations among these patients, a high percentage (29%) of FAP-associated desmoids showed loss of the APC region at 5q22.2, which was infrequently (3%) seen among sporadic tumours. Our data suggest that loss of region 6q15-q16.2 is an important event in FAP-associated as well as sporadic desmoids, most likely of relevance for desmoid tumour progression.     

Fast local fragment chaining using sum-of-pair gap costs

Background

When inferring phylogenetic trees different algorithms may give different trees. To study such effects a measure for the distance between two trees is useful. Quartet distance is one such measure, and is the number of quartet topologies that differ between two trees.

Results

We have derived a new algorithm for computing the quartet distance between a pair of general trees, i.e. trees where inner nodes can have any degree ≥ 3. The time and space complexity of our algorithm is sub-cubic in the number of leaves and does not depend on the degree of the inner nodes. This makes it the fastest algorithm so far for computing the quartet distance between general trees independent of the degree of the inner nodes.

Conclusions

We have implemented our algorithm and two of the best competitors. Our new algorithm is significantly faster than the competition and seems to run in close to quadratic time in practice.     

Semantic processing disturbance in patients with schizophrenia: a meta-analysis of the N400 component

Background

Theoretically semantic processing can be separated into early automatic semantic activation and late contextualization. Semantic processing deficits have been suggested in patients with schizophrenia, however it is not clear which stage of semantic processing is impaired. We attempted to clarify this issue by conducting a meta-analysis of the N400 component.

Methods

Twenty-one studies met the inclusion criteria for the meta-analysis procedure. The Comprehensive Meta-Analysis software package was used to compute pooled effect sizes and homogeneity.

Results

Studies favoring early automatic activation produced a significant effect size of −0.41 for the N400 effect. Studies favoring late contextualization generated a significant effect size of −0.36 for the N400 effect, a significant effect size of −0.52 for N400 for congruent/related target words, and a significant effect size of 0.82 for the N400 peak latency.

Conclusion

These findings suggest the automatic spreading activation process in patients with schizophrenia is very similar for closely related concepts and weakly or remotely related concepts, while late contextualization may be associated with impairments in processing semantically congruent context accompanied by slow processing speed.     

Comparative profiling of primary colorectal carcinomas and liver metastases identifies LEF1 as a prognostic biomarker

Purpose

We sought to identify genes of clinical significance to predict survival and the risk for colorectal liver metastasis (CLM), the most common site of metastasis from colorectal cancer (CRC).

Patients and Methods

We profiled gene expression in 31 specimens from primary CRC and 32 unmatched specimens of CLM, and performed Significance Analysis of Microarrays (SAM) to identify genes differentially expressed between these two groups. To characterize the clinical relevance of two highly-ranked differentially-expressed genes, we analyzed the expression of secreted phosphoprotein 1 (SPP1 or osteopontin) and lymphoid enhancer factor-1 (LEF1) by immunohistochemistry using a tissue microarray (TMA) representing an independent set of 154 patients with primary CRC.

Results

Supervised analysis using SAM identified 963 genes with significantly higher expression in CLM compared to primary CRC, with a false discovery rate of <0.5%. TMA analysis showed SPP1 and LEF1 protein overexpression in 60% and 44% of CRC cases, respectively. Subsequent occurrence of CLM was significantly correlated with the overexpression of LEF1 (chi-square p = 0.042), but not SPP1 (p = 0.14). Kaplan Meier analysis revealed significantly worse survival in patients with overexpression of LEF1 (p<0.01), but not SPP1 (p = 0.11). Both univariate and multivariate analyses identified stage (p<0.0001) and LEF1 overexpression (p<0.05) as important prognostic markers, but not tumor grade or SPP1.

Conclusion

Among genes differentially expressed between CLM and primary CRC, we demonstrate overexpression of LEF1 in primary CRC to be a prognostic factor for poor survival and increased risk for liver metastasis.