Neat plasma proteomics: getting the best out of the worst.

Plasma proteomics holds immense potential for clinical research and biomarker discovery, serving as a non-invasive "liquid biopsy" for tissue sampling. Mass spectrometry (MS)-based proteomics, thanks to improvement in speed and robustness, emerges as an ideal technology for exploring the plasma proteome for its unbiased and highly specific protein identification and quantification. Despite its potential, plasma proteomics is still a challenge due to the vast dynamic range of protein abundance, hindering the detection of less abundant proteins. Different approaches can help overcome this challenge. Conventional depletion methods face limitations in cost, throughput, accuracy, and off-target depletion. Nanoparticle-based enrichment shows promise in compressing dynamic range, but cost remains a constraint. Enrichment strategies for extracellular vesicles (EVs) can enhance plasma proteome coverage dramatically, but current methods are still too laborious for large series. Neat plasma remains popular for its cost-effectiveness, time efficiency, and low volume requirement. We used a test set of 33 plasma samples for all evaluations. Samples were digested using S-Trap and analyzed on Evosep One and nanoElute coupled to a timsTOF Pro using different elution gradients and ion mobility ranges. Data were mainly analyzed using library-free searches using DIA-NN. This study explores ways to improve proteome coverage in neat plasma both in MS data acquisition and MS data analysis. We demonstrate the value of sampling smaller hydrophilic peptides, increasing chromatographic separation, and using library-free searches. Additionally, we introduce the EV boost approach, that leverages on the extracellular vesicle fraction to enhance protein identification in neat plasma samples. Globally, our optimized analysis workflow allows the quantification of over 1000 proteins in neat plasma with a 24SPD throughput. We believe that these considerations can be of help independently of the LC-MS platform used.

Naive Pluripotent and Trophoblastic Stem Cell Lines as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project.

The Chromosome-centric Human Proteome Project (C-HPP) aims at identifying the proteins as gene products encoded by the human genome, characterizing their isoforms and functions. The existence of products has now been confirmed for 93.2% of the genes at the protein level. The remaining mostly correspond to proteins of low abundance or difficult to access. Over the past years, we have significantly contributed to the identification of missing proteins in the human spermatozoa. We pursue our search in the reproductive sphere with a focus on early human embryonic development. Pluripotent cells, developing into the fetus, and trophoblast cells, giving rise to the placenta, emerge during the first weeks. This emergence is a focus of scientists working in the field of reproduction, placentation and regenerative medicine. Most knowledge has been harnessed by transcriptomic analysis. Interestingly, some genes are uniquely expressed in those cells, giving the opportunity to uncover new proteins that might play a crucial role in setting up the molecular events underlying early embryonic development. Here, we analyzed naive pluripotent and trophoblastic stem cells and discovered 4 new missing proteins, thus contributing to the C-HPP. The mass spectrometry proteomics data was deposited on ProteomeXchange under the data set identifier PXD035768.

Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: Application to cerebrospinal fluid.

RATIONALE: Glycosylation is one of the most complex types of post-translational modifications of proteins. The alteration of glycans bound to proteins from cerebrospinal fluid (CSF) in relation to disorders of the central nervous system is a highly relevant subject, but only few studies have focused on the glycosylation of CSF proteins. METHODS: Reproducible profiles of CSF N-glycans were first obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after permethylation. Tryptic glycopeptides from CSF proteins were also enriched by hydrophilic interaction, and the resulting extracts divided into two equal aliquots. A first aliquot was enzymatically deglycosylated and analyzed by nano-liquid chromatography/tandem mass spectrometry while the second one, containing intact enriched glycopeptides, was directly analyzed. Site-specific data were obtained by combining the data from these three experiments. RESULTS: We describe the development of a versatile approach for obtaining site-specific information on the N-glycosylation of CSF glycoproteins. Under these conditions, 124 N-glycopeptides representing 55 N-glycosites from 36 glycoproteins were tentatively identified. Special emphasis was placed on the analysis of glycoproteins/glycopeptides bearing 'brain-type' N-glycans, representing potential biologically relevant structures in the field of neurodegenerative disorders. Using our workflow, only a few proteins were shown to carry such particular glycan motifs. CONCLUSIONS: We developed an approach combining N-glycomics and N-glycoproteomics and underline its usefulness to study the site-specific glycosylation of major human CSF proteins. The final rather long-term objective is to combine these data with those from other omics approaches to delve deeper into the understanding of particular neurological disorders.

Proteomic analysis of virus-host interactions in an infectious context using recombinant viruses.

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells.

Imaging mass spectrometry provides fingerprints for distinguishing hepatocellular carcinoma from cirrhosis.

MALDI imaging mass spectrometry (MALDI IMS) is a powerful tool for comprehending the spectrum of peptides/proteins expressed in tissue sections. The aim of the present study was to investigate, using MALDI IMS, the proteome of hepatocellular carcinomas (HCC) and to compare it with peritumoral cirrhosis so as to characterize new biomarkers of HCC. Frozen liver tissues corresponding to HCC and background cirrhosis (n = 30) were selected and subjected to MALDI IMS. We found a set of proteins/peptides with a differential intensity level that most accurately delineated cancer from adjacent cirrhotic tissue. Using a support vector machine algorithm, we generated a classification model in the train set that enabled segmenting images from the independent validation set and that in most cases matched histologic analysis. The most discriminating peak (m/z 8565) more intense in HCC was characterized as the monomeric ubiquitin. An immunohistochemical study in a large series of HCC/cirrhosis sampled on tissue microarray supported that ubiquitin was overexpressed in HCC. We demonstrated also that this increase was not related to an upregulation of ubiquitin gene transcription in HCC, thus suggesting a post-transcriptional mechanism. This approach might provide a new tool for diagnosis of difficult HCC cases and an opportunity for identifying candidate biomarkers.

In situ isobaric lipid mapping by MALDI-ion mobility separation-mass spectrometry imaging.

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high-resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.

Secretome analysis of Trypanosoma cruzi by proteomics studies.

BACKGROUND: Chagas disease is a debilitating often fatal disease resulting from infection by the protozoan parasite Trypanosoma cruzi. Chagas disease is endemic in 21 countries of the Americas, and it is an emerging disease in other countries as a result of migration. Given the chronic nature of the infection where intracellular parasites persist for years, the diagnosis of T. cruzi by direct detection is difficult, whereas serologic tests though sensitive may yield false-positive results. The development of new rapid test based on the identification of soluble parasitic antigens in serum would be a real innovation in the diagnosis of Chagas disease. METHODS: To identify new soluble biomarkers that may improve diagnostic tests, we investigated the proteins secreted by T. cruzi using mass spectrometric analyses of conditioned culture media devoid of serum collected during the emergence of trypomastigotes from infected Vero cells. In addition, we compared the secretomes of two T. cruzi strains from DTU Tc VI (VD and CL Brener). RESULTS: Analysis of the secretome collected during the emergence of trypomastigotes from Vero cells led to the identification of 591 T. cruzi proteins. Three hundred sixty three proteins are common to both strains and most belong to different multigenic super families (i.e. TcS, GP63, MASP, and DGF1). Ultimately we have established a list of 94 secreted proteins, common to both DTU Tc VI strains that do not belong to members of multigene families. CONCLUSIONS: This study provides the first comparative analysis of the secretomes from two distinct T. cruzi strains of DTU TcVI. This led us to identify a subset of common secreted proteins that could potentially serve as serum markers for T. cruzi infection. Their potential could now be evaluated, with specific antibodies using sera collected from patients and residents from endemic regions.

A new study of the bacterial lipidome: HPTLC-MALDI-TOF imaging enlightening the presence of phosphatidylcholine in airborne Pseudomonas fluorescens MFAF76a.

Lipids are major functional components of bacterial cells that play fundamental roles in bacterial metabolism and the barrier function between cells and the environment. In an effort to investigate the bacterial lipidome, we adopted a protocol using MALDI-TOF MS imaging coupled to HPTLC to screen a large number of phospholipid classes in a short span of time. With this method, phospholipids of airborne Pseudomonas fluorescens MFAF76a were visualized and identified in sample extracts (measurement accuracy below 0.1 Da, phospholipid identification by means of four characteristic fragment peaks). Via this technique, the P. fluorescens lipidome was shown to comprise three major lipid classes: phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The protocol described herein is simple, rapid and effective for screening of bacterial phospholipid classes. The remarkable presence of a eukaryotic phospholipid, phosphatidylcholine, was observed in P. fluorescens MFAF76a. This lipid is known to play a role in bacteria-host interactions and had not been known to be found in P. fluorescens cells.