Background Multiple sclerosis (MS) is an autoimmune, neuroinflammatory disease, with an unclear etiology. However, T cells play a central role in the pathogenesis by crossing the blood–brain-barrier, leading to inflammation of the central nervous system and demyelination of the protective sheath surrounding the nerve fibers. MS has a complex inheritance pattern, and several studies indicate that gene interactions with environmental factors contribute to disease onset. Methods In the current study, we evaluated T cell dysregulation at the protein level using electrospray liquid chromatography–tandem mass spectrometry to get novel insights into immune-cell processes in MS. We have analyzed the proteomic profiles of CD4 + and CD8 + T cells purified from whole blood from 13 newly diagnosed, treatment-naive female patients with relapsing–remitting MS and 14 age- and sex-matched healthy controls. Results An overall higher protein abundance was observed in both CD4 + and CD8 + T cells from MS patients when compared to healthy controls. The differentially expressed proteins were enriched for T-cell specific activation pathways, especially CTLA4 and CD28 signaling in CD4 + T cells. When selectively analyzing proteins expressed from the genes most proximal to > 200 non-HLA MS susceptibility polymorphisms, we observed differential expression of eight proteins in T cells between MS patients and healthy controls, and there was a correlation between the genotype at three MS genetic risk loci and protein expressed from proximal genes. Conclusion Our study provides evidence for proteomic differences in T cells from relapsing–remitting MS patients compared to healthy controls and also identifies dysregulation of proteins encoded from MS susceptibility genes. Electronic supplementary material The online version of this article (10.1186/s12014-019-9241-5) contains supplementary material, which is available to authorized users.
BackgroundSatisfactory sample preparation for mass spectrometry-based analysis is a critical step in the proteomics workflow. The quality and reproducibility of sample preparation can determine the coverage and confidence of proteomics results. Up to date, several methodologies have been described to produce suitable peptides for mass spectrometry analysis, followed by strategies for enrichment of post-translational modified peptides, if desired. Among them, the filter-aided sample preparation (FASP) has been introduced as a method to allow for removal of denaturants, reductants, alkylators, lipids and nucleic acids prior to trypsin digestion. Despite the high proteolytic digestion and contaminant removal efficiency described for this method, filter failure and consequently complete sample loss can discourage the use of this approach by the proteomic community.ResultsAs judged by our quality controls, we were able to perform reliable and reproducible FASP for mass spectrometry analysis that allowed the quantification of 2141 proteins and 3694 phosphopeptides from as little as 20 and 320 μg of protein lysate from acute myeloid leukemia (AML) patients, respectively. Using the immobilized metal ion affinity chromatography (IMAC) method resulted in samples specifically enriched in phosphopeptides and allowed the quantification of a high number of both di- and multi-phosphopeptides in addition to the abundant mono-phosphopeptides. The workflows’ high reproducibility from three biological replicates was demonstrated by the similar number of quantified proteins and localized phosphosites, and confirmed by the similar distributions of their molecular functions. We found that the combination of the FASP procedure with StageTip mixed-mode fractionation and IMAC are excellent workflows for the reproducible and deep study of AML proteomes and phosphoproteomes, respectively.ConclusionsThe FASP procedure can be carried out without the risk of filter failure by performing a simple test of the filter quality before adding the protein sample. Herein, we demonstrate an efficient and reproducible FASP-based pipeline for the proteomic and phosphoproteomic analysis of AML patient samples which also can be used for the analysis of any other protein samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0043-0) contains supplementary material, which is available to authorized users.
Abstract.A method for determination of the fatty acids of blubber fat from living seals is demonstrated: fat samples are retrieved by a small syringe, the fat is directly methanolysed, the resulting fatty acid methyl esters are gas-chromatographed and the chromatographic results are treated by multivariate principal-component analysis. The blubber fat of two grey seals [Halichoerus grypus (Fabricius, 1791)] and two harbor seals (Phoca vitulina L.) at the Aquarium of Bergen, Norway, as well as their diet, herring and mackerel, were monitored over a period of 7 mo in 1989. Fatty acid composition of the blubber fat was significantly different from that of the diet.
As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super-SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm-13, NB4, MV4-11, THP-1, and OCI-AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441.
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