BACKGROUND:Low-volume trapping columns are essential for sample enrichment, desalting, and injection profile focusing on nano-LC-MS-based proteomics. They enable higher sample loading, improve chromatographic performance, and protect the analytical column by removing salts and contaminants. Recently, monolithic trap columns with micropillar architecture have emerged as alternatives to conventionally packed traps. This study compares the performance of a packed and a micropillar monolithic trap column for the analysis of tryptic peptides. METHODS:A tryptic digest of HeLa cell lysate was analyzed under identical LC-MS conditions using both trap types. Peptides were detected at 214 nm and analyzed by nano-ESI on a Q Exactive Plus Orbitrap. Data were searched against the human UniProt database (February 2023) using FragPipe v20.0, and statistical evaluation of MaxLFQ intensities was performed in Perseus using Welch's t-test and clustering analysis. RESULTS:Over 2500 proteins were identified with both setups. The packed trap column yielded more total peptides, particularly those with post-translational modifications and higher hydrophilicity, whereas the monolithic column favored peptides of intermediate hydrophobicity. Chromatographic profiles confirmed a slight reduction in the trapping efficiency of hydrophilic peptides by the monolithic trap. CONCLUSIONS:Trap column design significantly influences peptide recovery and proteome coverage.

Abstract Metaproteomics measures functional expression in complex microbial communities, but extreme sample complexity and dynamic range challenge acquisition strategies. Trapped ion mobility spectrometry with parallel accumulation–serial fragmentation (PASEF) has expanded into multiple acquisition modes, yet systematic evaluations in high-complexity metaproteomes remain limited. Here, we benchmark five PASEF modes—DDA-, DIA-, Slice-, Synchro-, and midia-PASEF—using a complex fecal peptide background spiked with defined bacterial references. Across three gradients and input levels, 540 LC–MS acquisitions are analyzed under matched conditions. Based on data-derived performance scores, DIA-based strategies outperform DDA-PASEF in peptide and protein coverage, particularly for low-abundance microbial features. DIA- and Slice-PASEF show strong quantitative reproducibility, reduced ratio compression, and consistent species-abundance scaling, while functional profiling reveals expanded annotation depth. When tested in a murine colonic injury model, the two highest-scoring methods, DIA- and Slice-PASEF, capture concordant host and microbial responses.

Merkel cell carcinomas (MCCs) are rare, highly aggressive skin cancers with poor outcome due to early lymphatic tumor spread and frequent recurrences. MCCs mostly occur in the head and neck and are mainly caused by an infection with the Merkel cell polyomavirus (MCPyV). Increasing evidence suggests that Piwil-2 and small non-coding PIWI-interacting RNAs (piRNAs) play an important role in solid malignancies and we thought that this might also be the case for MCCs. Therefore, Piwil-2 expression was first evaluated in 27 MCC specimens and correlated with oncological outcome. We found an association with high Piwil-2 expression and advanced tumor stage, MCPyV positivity and poor outcome. Next, we utilized siRNAs for Piwil-2 knock-down in MCC cells. Downregulation of Piwil-2 caused a significant change of 202 different piRNAs and 419 proteins. Interestingly, proteins related to viral driven MCC pathways (TRRAP, BRD8, PRIM2, ORC4) were significantly downregulated. Moreover, there was a moderate cell cycle arrest of cells in the G0/G1-phase, as well as a significant upregulation of SOX-2, a key regulator of Merkel cells. Altogether, Piwil-2 poses a poor prognosticator in MCCs, which is linked to MCC oncogenesis and SOX-2. Further research is needed to better understand underlying mechanisms and to prove their clinical relevance.

It is becoming increasingly important to use high-throughput analysis of clinical samples to monitor the progression or regression of disease or the concentration of therapeutic agents during personalized treatment. The use of micro and capillary chromatography is well established, but nano-LC can provide significantly higher sensitivity, especially for analyzing therapeutic monoclonal antibodies. Although the nano-LC has been considered a slow procedure, new separation columns, for example, monolithic columns, can provide both high sample throughput and high sensitivity. When hyphenated with a high-end mass spectrometer, the combination can be used for screening and targeted analyses of clinical samples. Furthermore, a combination of different, orthogonal separation methods can be used to increase the selectivity and sensitivity of the analysis. Some actual research and applied topics for clinical sample analyses will be discussed.

BackgroundHuman induced pluripotent stem cells represent a scalable source of youthful tissue progenitors and secretomes for regenerative therapies. The aim of our study was to investigate the potential of conditioned medium (CM) from hiPSC-mesenchymal progenitors (hiPSC-MPs) to stimulate osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (MSCs). We also investigated whether prolonged cultivation or osteogenic pre-differentiation of hiPSC-MPs could enhance the stimulatory activity of CM.MethodsMSCs were isolated from 13 donors (age 20-90 years). CM derived from hiPSC-MPs was added to the MSC cultures and the effects on proliferation and osteogenic differentiation were examined after 14 days and 6 weeks. The stimulatory activity of hiPSC-MP-CM was compared with the activity of MSC-derived CM and with the activity of CM prepared from hiPSC-MPs pre-cultured in growth or osteogenic medium for 14 days. Comparative proteomic analysis of CM was performed to gain insight into the molecular components responsible for the stimulatory activity.ResultsPrimary bone marrow-derived MSC exhibited variability, with a tendency towards lower proliferation and tri-lineage differentiation in older donors. hiPSC-MP-CM increased the proliferation and alkaline phosphatase activity of MSC from several adult/aged donors after 14 days of continuous supplementation under osteogenic conditions. However, CM supplementation failed to improve the mineralization of MSC pellets after 6 weeks under osteogenic conditions. hiPSC-MP-CM showed greater enhancement of proliferation and ALP activity than CM derived from bone marrow-derived MSCs. Moreover, 14-day cultivation but not osteogenic pre-differentiation of hiPSC-MPs strongly enhanced CM stimulatory activity. Quantitative proteomic analysis of d14-CM revealed a distinct profile of components that formed a highly interconnected associations network with two clusters, one functionally associated with binding and organization of actin/cytoskeletal components and the other with structural constituents of the extracellular matrix, collagen, and growth factor binding. Several hub proteins were identified that were reported to have functions in cell-extracellular matrix interaction, osteogenic differentiation and development.ConclusionsOur data show that hiPSC-MP-CM enhances early osteogenic differentiation of human bone marrow-derived MSCs and that prolonged cultivation of hiPSC-MPs enhances CM-stimulatory activity. Proteomic analysis of the upregulated protein components provides the basis for further optimization of hiPSC-MP-CM for bone regenerative therapies.

BackgroundEfforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype.MethodsWe performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from 7 patients and further integrated these data with a cohort of EAC RNA-seq data (n=264 patients), EAC whole-genome sequencing (n=454 patients) and external published datasets.Central findingsWe quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated Protein and RNA abundance, suggesting post-transcriptional regulation of protein expression. These outlier genes including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be over-expressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed.ConclusionsBy quantitative proteomic analysis we have identified GPA33 as an EAC-selective biomarker. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, post-transcriptional regulation of protein expression which may be an exploitable vulnerability.

Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disorder characterized by bone fragility and reduced bone mass generally caused by defects in type I collagen structure or defects in proteins interacting with collagen processing. We identified a homozygous missense mutation in SEC16B in a child with vertebral fractures, leg bowing, short stature, muscular hypotonia, and bone densitometric and histomorphometric features in keeping with OI with distinct ultrastructural features. In line with the putative function of SEC16B as a regulator of trafficking between the ER and the Golgi complex, we showed that patient fibroblasts accumulated type I procollagen in the ER and exhibited a general trafficking defect at the level of the ER. Consequently, patient fibroblasts exhibited ER stress, enhanced autophagosome formation, and higher levels of apoptosis. Transfection of wild-type SEC16B into patient cells rescued the collagen trafficking. Mechanistically, we show that the defect is a consequence of reduced SEC16B expression, rather than due to alterations in protein function. These data suggest SEC16B as a recessive candidate gene for OI.

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

DEL-1 (developmental endothelial locus-1) induces integrin signaling and recognizes phosphatidylserine exposed on apoptotic cells. We show that DEL-1, which is thought to be a secreted molecule, is not found in melanoma cell culture supernatants but is exported by an endosomal pathway and released via small extracellular vesicles (sEV). Proteomics of DEL-1 positive sEV, but not of DEL-1 negative sEV contain proteins associated with poor survival in cancer. To determine whether DEL-1 is suitable to predict treatment responses, we isolated sEV from plasma of melanoma patients before and 90 days of treatment with checkpoint inhibitors. Although we could not detect DEL-1 in plasma sEV even in patients with progressive disease (most likely due to the very low protein yield from sEV isolated from 1ml plasma), the principal component analysis allowed a clear differentiation between controls and patients as well as between patients before and after treatment. Interestingly, in one patient with complete regression, in the post treatment sample, the protein expression profile remained in the pre-treatment cluster. The low protein yield of patient sEV and the low patient number are clear limitations of this study, but results demonstrate that this method could have the potential to predict treatment responses.

Background Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome. Differences in transcript and the corresponding protein abundances remain under-explored, leaving gaps in our understanding of the mechanisms underlying the disease. Methods We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance used tissue-matched RNAseq and proteomic data from 7 patients and further integrated these data with a cohort of EAC RNA-seq data (n=264 patients), whole-genome sequencing (n=454 patients) and external published datasets. Results We quantified protein expression from 5897 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-specific expression were identified including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated Protein and RNA abundance, suggesting post-transcriptional regulation of protein expression. These outlier genes including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be over-expressed at the protein level in EAC compared to adjacent normal tissues with an EAC-specific post-transcriptional mechanism of regulation of protein expression proposed. Conclusions By quantitative proteomic analysis we have identified GPA33 as an EAC-specific biomarker. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-specific post-transcriptional regulation of protein expression which may be an exploitable vulnerability.

Die Molaren-Inzisiven-Hypomineralisation (MIH) stellt eine entwicklungsbedingte Erkrankung der Zahnhartsubstanz dar, deren Ätiologie nicht geklärt ist. In Anbetracht der Tatsache, dass Speichel die Schmelzremineralisation kontrolliert und dass die MIH mit einer höheren Speichelflussrate assoziiert ist, wurde die Hypothese aufgestellt, dass die Proteinzusammensetzung des Speichels mit der Erkrankung in Zusammenhang steht. Um dies zu testen, wurden 5 kariesfreie Kinder im Alter von 6-14 Jahren mit einer MIH und 5 kariesfreie Kinder ohne MIH in die Studie eingeschlossen und Speichelproben entnommen. Diese wurden anschließend einer Proteomanalyse unterzogen, gefolgt von einer Proteinklassifizierung in biologische Signalwege. Von 618 Speichelproteinen, die identifiziert wurden, wurden 88 Proteine ausschließlich bei MIH-Patienten und 16 Proteine nur bei Kindern ohne MIH erkannt. Die Analyse der biologischen Signalwege zeigte, dass diese 88 Proteine bei den MIH-Patienten mit der von neutrophilen Granulozyten-vermittelten adaptiven Immunität, der Aktivierung des klassischen Signalwegs der Komplementaktivierung, dem Abbau der extrazellulären Matrix, dem Häm-Scaveng- sowie dem Glutathion- und dem Medikamentenmetabolismus assoziiert sind. Die Ergebnisse deuten darauf hin, dass die Proteinzusammensetzung des Speichels bei MIH-Patienten ein kataboles Milieu widerspiegelt, welches mit Entzündungen verbunden ist.

Objective To report an unusual clinical phenotype of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis and describe associated neuropathologic findings. Methods We retrospectively investigated 3 AMPAR encephalitis patients with autoimmune global hippocampal amnesia using comprehensive cognitive and neuropsychologic assessment, antibody testing by in-house tissue-based and cell-based assays, and neuropathologic analysis of brain autopsy tissue including histology and immunohistochemistry. Results Three patients presented with acute-to-subacute global amnesia without affection of cognitive performance, attention, concentration, or verbal function. None of the patients had epileptic seizures, change of behavior, personality changes, or psychiatric symptoms. The MRI was normal in 1 patient and showed increased fluid-attenuated inversion recovery/T2 signal in the hippocampus in the other 2 patients. Two patients showed complete remission after immunotherapy. The one patient who did not improve had an underlying adenocarcinoma of the lung and died 3.5 months after disease onset because of tumor progression. Neuropathologic analysis of the brain autopsy revealed unilateral hippocampal sclerosis accompanied by mild inflammatory infiltrates, predominantly composed of T lymphocytes, and decrease of AMPAR immunoreactivity. Conclusion AMPAR antibodies usually associate with limbic encephalitis but may also present with immune responsive, acute-to-subacute, isolated hippocampal dysfunction without overt inflammatory CSF or MRI changes.

Nanophthalmos-4 is a rare autosomal dominant disorder caused by two known variations in TMEM98. An Austrian Caucasian pedigree was identified suffering from nanophthalmos and late onset angle-closure glaucoma and premature loss of visual acuity. Whole exome sequencing identified segregation of a c.602G > C transversion in TMEM98 (p.Arg201Pro) as potentially causative. A protein homology model generated showed a TMEM98 structure comprising α4, α5/6, α7 and α8 antiparallel helix bundles and two predicted transmembrane domains in α1 and α7 that have been confirmed in vitro. Both p.Arg201Pro and the two missense variations representing proline insertions identified previously to cause nanophthalmos-4 (p.Ala193Pro and p.His196Pro) are located in the charge polarized helix α8 (p.183-p210). Stability of the C-terminal alpha helical structure of TMEM98 is therefore essential to prevent the development of human nanophthalmos-4. Precise molecular diagnosis could lead to the development of tailored therapies for patients with orphan ocular disease.

Particulate autologous tooth roots are increasingly used for alveolar bone augmentation; however, the proteomic profile of acid dentin lysate and the respective cellular response have not been investigated. Here we show that TGF-β1 is among the 226 proteins of acid dentin lysate (ADL) prepared from porcine teeth. RNA sequencing identified 231 strongly regulated genes when gingival fibroblasts were exposed to ADL. Out of these genes, about one third required activation of the TGF-β receptor type I kinase including interleukin 11 (IL11) and NADPH oxidase 4 (NOX4). Reverse transcription-quantitative polymerase chain reaction and immunoassay confirmed the TGF-β-dependent expression of IL11 and NOX4. The activation of canonical TGF-β signaling by ADL was further confirmed by the phosphorylation of Smad3 and translocation of Smad2/3, using Western blot and immunofluorescence staining, respectively. Finally, we showed that TGF-β activity released from dentin by acid lysis adsorbs to titanium and collagen membranes. These findings suggest that dentin particles are a rich source of TGF-β causing a major response of gingival fibroblasts.

Despite an estimated prevalence of 13% in women, the exact etiology of non-neurogenic overactive bladder syndrome is unclear. The aim of our study was to gain a better understanding of the pathophysiology of female overactive bladder syndrome by mapping the urinary proteomic profile. We collected urine samples of 20 patients with overactive bladder syndrome and of 20 controls. We used mass spectrometric analysis for label-free quantitation, Swissprot human database for data search, Scaffold for data allocation and the Reactome Knowledgebase for final pathway enrichment analysis. We identified 1897 proteins at a false discovery rate of 1% and significance level p < 0.001. Thirty-seven significant proteins of the case group and 53 of the control group met the criteria for further pathway analysis (p < 0.0003 and Log2 (fold change) >2). Significant proteins of the overactive bladder group were, according to the 25 most relevant pathways, mainly involved in cellular response to stress and apoptosis. In the control group, significant pathways mainly concerned immunological, microbial-protective processes and tissue- elasticity processes. These findings may suggest a loss of protective factors as well as increased cellular response to stress and apoptosis in overactive bladder syndrome.

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-β1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-β1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-β receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-β1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-β receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.

In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient’s course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections.