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The rapidly increasing adoption of high-resolution accurate-mass methods in analytical laboratories has fueled demand for instruments that combine high performance and reliability with small size and greater ease-of-use. This paper presents the major design principles that are driving the evolution of the hybrid quadrupole-Orbitrap instrument architecture to enable a greater range of applications and users. These principles may be summarized as follows: better usage of physical space and better access for service by means of size reduction of pumping and ion optics; expanded use of technologies from electronics in ion-optical design; flexibility in performance via modularity of design of the hardware and software components; and, harmonization of interfaces with other instruments to facilitate sharing and transferability of analytical workflows. The design of a novel family of hybrid mass spectrometers is described in detail, and performance evaluation is carried out on a wide variety of samples for its three representatives: the Orbitrap Exploris 120, Orbitrap Exploris 240 and Orbitrap Exploris 480 mass spectrometers. The new instrument family is shown to offer compelling potential not only for high-end proteomics and biopharmaceutical applications, but also for screening, trace, targeted and clinical analysis by liquid chromatography/mass spectrometry methods.

Although current LC-MS technology permits scientists to efficiently screen clinical samples in translational research, e.g., steroids, biogenic amines, and even plasma or serum proteomes, in a daily routine, maintaining the balance between throughput and analytical depth is still a limiting factor. A typical approach to enhance the proteome depth is employing offline two-dimensional (2D) fractionation techniques before reversed-phase nanoLC-MS/MS analysis (1D-nanoLC-MS). These additional sample preparation steps usually require extensive sample manipulation, which could result in sample alteration and sample loss. Here, we present and compare 1D-nanoLC-MS with an automated online-2D high-pH RP × low pH RP separation method for deep proteome profiling using a nanoLC system coupled to a high-resolution accurate-mass mass spectrometer. The proof-of-principle study permitted the identification of ca. 500 proteins with ∼10,000 peptides in 15 enzymatically digested crude serum samples collected from healthy donors in 3 laboratories across Europe. The developed method identified 60% more peptides in comparison with conventional 1D nanoLC-MS/MS analysis with ca. 4 times lower throughput while retaining the quantitative information. Serum sample preparation related changes were revealed by applying unsupervised classification techniques and, therefore, must be taken into account while planning multicentric biomarker discovery and validation studies. Overall, this novel method reduces sample complexity and boosts the number of peptide and protein identifications without the need for extra sample handling procedures for samples equivalent to less than 1 μL of blood, which expands the space for potential biomarker discovery by looking deeper into the composition of biofluids.

Orbitrap Exploris 480 MS with FAIMS Pro provides fast, sensitive and robust profiling of proteomes when combined with Evosep One. The combination of Data Independent Acquisition (DIA) and FAIMS with single compensation voltages enables analysis of up to 2000 peptides per LC gradient minute and more than 5000 protein groups in twenty minutes. DIA-FAIMS based label-free quantitation achieves similar depth and quantitative accuracy as the well-established isobaric labelling approaches, but with less LC-MS measurement time, making it a good choice for large cohort of samples., Graphical Abstract Highlights Increased proteome coverage with Orbitrap Exploris 480 MS and FAIMS using single compensation voltages and short LC gradients. Towards single-cell proteomics with high-sensitivity analysis of 5 ng HeLa with more than 1,000 proteins identified in 5 minutes using FAIMS and DIA. Deep proteome profiling across twelve rat organs tissues by label-free quantitation using DIA compared to TMT-multiplexing and turboTMT acquisition using phi-SDM. Rapid and sensitive phosphoproteomics with automated enrichment using Ti-IMAC magnetic beads and direct DIA analysis., State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

We have developed a new method for the identification of bacterial species causing Urinary Tract Infections. The first training step used DIA analysis on multiple replicates of bacterial inoculates to define a peptide signature by machine learning classifiers. In a second identification step, the signature is monitored by targeted proteomics on unknown samples. This fast, culture-free and accurate method paves the way of the development of new diagnostic approaches limiting the emergence of antimicrobial resistances., Graphical Abstract Highlights Fast and culture-free method for the identification of the 15 bacterial species causing UTIs. Combination of DIA analysis and machine learning algorithms to define a peptide signature. High accuracy, good linearity and reproducibility, sensitivity below standard threshold. Transferability to other laboratories and other mass spectrometers., Fast identification of microbial species in clinical samples is essential to provide an appropriate antibiotherapy to the patient and reduce the prescription of broad-spectrum antimicrobials leading to antibioresistances. MALDI-TOF-MS technology has become a tool of choice for microbial identification but has several drawbacks: it requires a long step of bacterial culture before analysis (≥24 h), has a low specificity and is not quantitative. We developed a new strategy for identifying bacterial species in urine using specific LC-MS/MS peptidic signatures. In the first training step, libraries of peptides are obtained on pure bacterial colonies in DDA mode, their detection in urine is then verified in DIA mode, followed by the use of machine learning classifiers (NaiveBayes, BayesNet and Hoeffding tree) to define a peptidic signature to distinguish each bacterial species from the others. Then, in the second step, this signature is monitored in unknown urine samples using targeted proteomics. This method, allowing bacterial identification in less than 4 h, has been applied to fifteen species representing 84% of all Urinary Tract Infections. More than 31,000 peptides in 190 samples were quantified by DIA and classified by machine learning to determine an 82 peptides signature and build a prediction model. This signature was validated for its use in routine using Parallel Reaction Monitoring on two different instruments. Linearity and reproducibility of the method were demonstrated as well as its accuracy on donor specimens. Within 4h and without bacterial culture, our method was able to predict the predominant bacteria infecting a sample in 97% of cases and 100% above the standard threshold. This work demonstrates the efficiency of our method for the rapid and specific identification of the bacterial species causing UTI and could be extended in the future to other biological specimens and to bacteria having specific virulence or resistance factors.

The identification of microbial species in biological samples is essential to many applications in health, food safety and environment. MALDI-TOF MS technology has become a tool of choice for microbial identification but it has several drawbacks including: it requires a long step of bacterial culture prior to analysis (24h), it has a low specificity and is not quantitative. We have developed a new strategy for identifying bacterial species in biological samples using specific LC-MS/MS peptidic signatures. In the first training step, deep proteome coverage of bacteria of interest is obtained in Data Independent Acquisition (DIA) mode, followed by the use of machine learning to define the peptides the most susceptible to distinguish each bacterial species from the others. Then, in the second step, this peptidic signature is monitored in biological samples using targeted proteomics. This method, which allows the bacterial identification from clinical specimens in less than 4h, has been applied to fifteen species representing 84% of all Urinary Tract Infections (UTI). More than 31000 peptides in 200 samples have been quantified by DIA and analyzed by machine learning to determine an 82 peptides signature and build prediction models able to classify the fifteen bacterial species. This peptidic signature was validated for its use in routine conditions using Parallel Reaction Monitoring on a capillary flow chromatography coupled to a Thermo Scientific™ Q Exactive HF-X instrument. Linearity and reproducibility of the method were demonstrated as well as its accuracy on donor specimens. Within 4h and without bacterial culture, our method was able to predict the predominant bacteria infecting a sample in 97% of cases and 100% above the 1×105 CFU/mL threshold commonly used by clinical laboratories. This work demonstrates the efficiency of our method for the rapid and specific identification of the bacterial species causing UTI and could be extended in the future to other biological specimens and to bacteria having specific virulence or resistance factors.

Aberrant neuronal network activity associated with neuronal hyperexcitability seems to be an important cause of cognitive decline in aging and Alzheimer’s disease (AD). Out of many antiepileptics, only levetiracetam improved cognitive dysfunction in AD patients and AD animal models by reducing hyperexcitability. As impaired inhibitory interneuronal function, rather than overactive neurons, seems to be the underlying cause, improving impaired neuronal function rather than quieting overactive neurons might be relevant in explaining the lack of activity of the other antiepileptics. Interestingly, improvement of cognitive deficits by levetiracetam caused by small levels of soluble Aβ was accompanied by improvement of synaptic function and plasticity. As the negative effects of Aβ on synaptic plasticity strongly correlate with mitochondrial dysfunction, wehypothesized that the effect of levetiracetam on synaptic activity might be raised by an improved mitochondrial function. Accordingly, we investigated possible effects of levetiracetam on neuronal deficits associated with mitochondrial dysfunction linked to aging and AD. Levetiracetam improved several aspects of mitochondrial dysfunction including alterations of fission and fusion balance in a cell model for aging and early late-onset AD. We demonstrate for the first time, using immunohistochemistry and proteomics, that the synaptic vesicle protein 2A (SV2a), the molecular target of levetiracetam, is expressed in mitochondria. In addition, levetiracetam shows significant effect on the opening of the mitochondrial permeability transition pore. Importantly, the effects of levetiracetam were significantly abolished when SV2a was knockdown using siRNA. In conclusion, interfering with the SV2a protein at the mitochondrial level and thereby improving mitochondrial function might represent an additional therapeutic effect of levetiracetam to improve symptoms of late-onset AD.

Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate the faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange data set PXD001305). We find the ultra-high-field Orbitrap mass analyzer to be capable of attaining a sequencing speed above 20 Hz, and it routinely exceeds 10 peptide spectrum matches per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from 1 μg of HeLa digest using a 1 h gradient, which is an approximately 30% improvement compared to that with previous instrumentation. In addition, we show that very deep proteome coverage can be achieved in less than 24 h of analysis time by offline high-pH reversed-phase peptide fractionation, from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multiday, multienzyme efforts. Finally, the acquisition methods are evaluated for single-shot phosphoproteomics, where we identify 7600 unique HeLa phosphopeptides in one gradient hour and find the quality of fragmentation spectra to be more important than quantity for accurate site assignment.

The biodistribution of nanoparticles is significantly influenced by their interaction with plasma proteins. In order to optimize and possibly monitor the delivery of drugs bound to nanoparticles across the blood–brain barrier (BBB), the protein adsorption pattern of uncoated poly(lactic-co-glycolic acid) (PLGA) nanoparticles after their incubation in human plasma was studied by mass spectrometry. After washing of the particles with water, the proteins were directly digested on the nanoparticle surface using trypsin and then analyzed by nLC MALDI-TOF/TOF. Up to now, the standard method for investigation into the plasma protein adsorption to the particles was 2D gel electrophoresis (2D-PAGE), in certain cases followed by mass spectrometry. The non-gel-based method proposed in the present study provides novel insights into the protein corona surrounding the nanoparticles. The proteins adsorbed on the PLGA nanoparticles after incubation that gave the best signal in terms of quality (high MASCOT score) in human plasma were apolipoprotein E, vitronectin, histidine-rich glycoprotein and kininogen-1. These proteins also are constituents of HDL.

In a preliminary test, four single hairs of a drug abuser were analyzed for the presence of drugs by MALDI Fourier transform mass spectrometry (MALDI-FTMS). Washed hair strains were directly fixed on a sample plate using pressure stable, double-sided adhesive tape; α-cyano-4-hydroxycinnamic acid matrix was manually spotted onto the hair strains. FTMS full scans were obtained moving from the hair root region towards the hair tip. Cocaine (accurate m/z ratio 304.15433) was identified mostly from the root of the hair and then later again towards the hair tip. This was confirmed by analysis of a second hair. Additionally cocaine metabolites with m/z ratio 290.13868 (benzoylecgonine), and m/z 318.16998 (cocaethylene) were detected for plausibility control. Using the MALDI technique, time-related information was obtained concerning the behavioural pattern of the consumer with high resolution compared to conventional procedures. However, in two hairs of the same individual which were analyzed under the same conditions, negative results were achieved. These preliminary results confirm the applicability of MALDI-MS for the determination of drugs and pharmaceuticals in hair samples being useful in forensic toxicology. The high chronological resolution allows an enhanced interpretation concerning the periods of drug administration. However, the negative results with two negative hairs have also demonstrated that hair analysis of single hairs can lead to misinterpretation. Different growth rates have to be considered, and particularly the phenomenon of different growth phases (anagen, catagen, telogen) require attention.

A simple, sensitive and fast method for the determination of melamine and its derivatives in milk powder using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed. Neither time-consuming sample preparation, nor special target plates, or other extra equipment are necessary. The common matrix sinapinic acid (SA) was used with a dried-droplet preparation. Detection limits (signal-to-noise (S/N) ratio = 3) for standard solutions of melamine, ammeline and cyanuric acid were 10, 25 and 10 µg/L, respectively. The limit of quantification (LOQ) for melamine was 25 µg/L and excellent linearity (R2: 0.9990) was maintained over the range of 10–2000 µg/L. Ammeline and cyanuric acid were analyzed with an LOQ of 50 µg/L and also excellent linearity (R2: 0.9997 and R2: 0.9998). Good accuracy and precision were obtained for all concentrations within the range of the standard curve. The developed method was successfully used for the determination of melamine, ammeline and cyanuric acid in milk powder samples with a simple sample preparation. The LOQ of melamine was 0.5 µg/g. Ammeline and cyanuric acid were detectable at 0.5 and 5 µg/g. This method showed excellent accuracy, precision and linearity and significantly reduces the needed analysis time, as only approximately 10 s/sample measuring time is required. To the authors' knowledge, this is the first published method to quantify melamine and derivatives by MALDI-TOF-MS. Copyright © 2011 John Wiley & Sons, Ltd.

Adipose tissue is an endocrine organ secreting different types of proteins, known as adipokines. These adipokines play important roles in homeostasis and metabolism. Adipocyte differentiation leads to a change in adipokine secretion profile which is probably involved in disruption of homeostasis. Many adipokines have been identified but species differences and limitations of human adipose tissue material urged the need for better model systems. Here we used a human cell strain derived from a Simpson Golabi Behmel syndrome (SGBS) patient. SGBS cells have already been used in functional studies on adipocytes but not in a proteomic search for adipokines. In this study, 2D-MS/MS and nLC-MALDI-MS/MS were applied to investigate secretion profiles of SGBS adipokines. A total of 80 secreted proteins were identified; 6 proteins are novel adipocyte secreted proteins, 20 proteins have not been detected before in human adipose material and 23 additional proteins previously detected in visceral adipose tissue have been found here secreted by SGBS-cells of subcutaneous origin. It can be concluded that SGBS cells are both a valid human cell model for adipocyte secretion profiling and for searching for novel human (pre)adipocytes secreted proteins.

Liquid chromatography, coupled with tandem mass spectrometry, is an established method for the identification of proteins from a complex sample. Despite its wide application, the analysis of whole proteomes still represents a challenge to researchers, because of the complexity and dynamic range of protein concentrations in biological samples. The analysis of such samples can be improved by adding a prefractionation step or a combination of orthogonal separation techniques. Off-gel isoelectric focusing (OGE) has successfully been used for prefractionation of a tryptic digest prior to nLC separation. In contrast to previous published results, we present a complete glycerol-free OGE for the analysis of purple membranes and Corynebacterium glutamicum membranes using the less-specific enzyme elastase. More than 85% of the identified unique peptides were found in solely one fraction, with very little carryover. These results are in accordance with those published for tryptic peptides. Therefore, OGE can be used as an effective prefractionation method in a multidimensional separation experiment of nontryptic membrane peptides.

Despite many advances in membrane proteomics during the last decade the fundamental problem of accessing the transmembrane regions itself has only been addressed to some extent. The present study establishes a method for the nano-LC-based analysis of complex membrane proteomes on the basis of a methanolic porcine pancreatic elastase digest to increase transmembrane coverage. Halobacterium salinarium purple and Corynebacterium glutamicum membranes were successfully analyzed by using the new protocol. We demonstrated that elastase digests yield a large proportion of transmembrane peptides, facilitating membrane protein identification. The potential for characterization of a membrane protein through full sequence coverage using elastase is there but is restricted to the higher abundance protein components. Compatibility of the work flow with the two most common mass spectrometric ionization techniques, ESI and MALDI, was shown. Currently better results are obtained using ESI mainly because of the low response of MALDI for strictly neutral peptides. New findings concerning elastase specificity in complex protein mixtures reveal a new prospect beyond the application in shotgun experiments. Furthermore peptide mass fingerprinting with less specific enzymes might be done in the near future but requires an adaptation of current search algorithms to the new proteases.

The hallmarks of Alzheimer’s disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network., Author Summary More than 20 years ago, the amyloid precursor protein (APP) was identified as the precursor protein of the Aβ peptide, the main component of senile plaques in brains affected by Alzheimer’s disease. However, little is known about the physiological function of amyloid precursor protein. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone highlights APP as a hitherto unknown player within the presynaptic network. The hippocampus is the most prominent brain region for learning and memory consolidation, and a vulnerable target for neurodegenerative disease, e. g. Alzheimer’s disease. Therefore, our experimental design is focused on the hippocampal neurotransmitter release site. Currently, the underlying mechanism of how APP acts within presynaptic networks is still elusive. Within the scope of this research article, we constructed a network of APP within the presynaptic active zone and how deletion of APP affects these individual networks. We combine bioinformatics tools and biochemical approaches to address the dataset provided by proteomics. Furthermore, we could unravel that APP executes regulatory functions within the synaptic vesicle cycle, cytoskeletal rearrangements and Ca2+-homeostasis. Taken together, our findings offer a new perspective on the physiological function of APP in the central nervous system and may provide a molecular link to the pathogenesis of Alzheimer’s disease.

Owing to its broad biological significance, the large-scale analysis of protein phosphorylation is more and more getting into the focus of proteomic research. Thousands of phosphopeptides can nowadays be identified using state-of-the-art tandem mass spectrometers in conjunction with sequence database searching, but localizing the phosphate group to a particular amino acid in the peptide sequence is often still difficult. Using 180 individually synthesized phosphopeptides with precisely known phosphorylation sites (p-sites), we have assessed the merits of the Mascot Delta Score (MD score) for the assignment of phosphorylation sites from tandem mass spectra (MS/MS) generated on four different matrix-assisted laser desorption ionization (MALDI) mass spectrometers including tandem time-of-flight (TOF/TOF), quadrupole time-of-flight, and ion trap mass analyzers. The results show that phosphorylation site identification is generally possible with false localization rates of about 10%. However, a comparison to previous work also revealed that phosphorylation site determination by MALDI MS/MS is less accurate than by ESI-MS/MS particularly if several and/or adjacent possible phosphorylation acceptor sites exist in a peptide sequence. We are making the tandem MS spectra and phosphopeptide collection available to the community so that scientists may adapt the MD scores reported here to their analytical environment and so that informatics developers may integrate the MD score into proteomic data analysis pipelines. peerReviewed

Detergents are frequently used for the solubilization of membrane proteins during and after purification steps. Unfortunately some of these detergents impair chromatographic separations and mass spectrometry (MS) analysis. Perfusion reversed-phase high-performance liquid chromatography (RP–HPLC) using POROS materials is suited for separating intact proteins solubilized by detergents due to the particles’ highly diffusive pores and chemical stability. In this article, the use of perfusive reversed-phase material packed into small inner diameter capillary columns is presented as a cheap, rapid, and efficient method for the removal of different types of detergents from protein solutions. The ability to purify and separate the subunits of membrane protein complexes with self-packed capillary columns is exemplified for bovine cytochrome bc 1 complex. Even highly hydrophobic subunits can be detected in collected fractions by intact mass measurements and identified after proteolytic digestion and matrix-assisted laser desorption/ionization tandem MS (MALDI MS/MS). The comparison with a gel-based approach shows that this method is a valuable alternative for purification and separation of intact proteins with subsequent MS analysis and that hydrophobic proteins are even better represented in the LC-based approach.

A simple, sensitive and fast method for the determination of melamine and its derivatives in milk powder using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed. Neither time-consuming sample preparation, nor special target plates, or other extra equipment are necessary. The common matrix sinapinic acid (SA) was used with a dried-droplet preparation. Detection limits (signal-to-noise (S/N) ratio = 3) for standard solutions of melamine, ammeline and cyanuric acid were 10, 25 and 10 µg/L, respectively. The limit of quantification (LOQ) for melamine was 25 µg/L and excellent linearity (R(2): 0.9990) was maintained over the range of 10-2000 µg/L. Ammeline and cyanuric acid were analyzed with an LOQ of 50 µg/L and also excellent linearity (R(2): 0.9997 and R(2): 0.9998). Good accuracy and precision were obtained for all concentrations within the range of the standard curve. The developed method was successfully used for the determination of melamine, ammeline and cyanuric acid in milk powder samples with a simple sample preparation. The LOQ of melamine was 0.5 µg/g. Ammeline and cyanuric acid were detectable at 0.5 and 5 µg/g. This method showed excellent accuracy, precision and linearity and significantly reduces the needed analysis time, as only approximately 10 s/sample measuring time is required. To the authors' knowledge, this is the first published method to quantify melamine and derivatives by MALDI-TOF-MS.

The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.

Expression of the AF4-MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4-MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4-MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4(me3) and H3K79(me3) histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.

The applicability of the less specific protease elastase for the identification of membrane and cytosolic proteins has already been demonstrated. MALDI as ionization technique particularly favors the detection of basic and to a lesser extent of weakly acidic peptides, whereas neutral peptides often remain undetected. Moreover, peptides below 700 Da are routinely excluded. In the following study, the advantage of additional information gained from tandem mass tag zero labeled peptides and the resultant increase in sequence coverage was evaluated. Through derivatization with tandem mass tag reagents, peptide measurement within the standard mass range of the MALDI reflector mode is achievable due to the mass increase. Compared to the unlabeled sample, peptides exhibiting relatively low molecular masses, pI values or higher hydrophobicity could be identified.

In the proteomic analysis of membrane proteins, less-specific proteases have become a promising tool to overcome fundamental limitations of trypsin with its unique specificity for basic residues. Pepsin is well-known to be utilized for specific applications that require acidic conditions, but in terms of membrane protein identification and characterization, it has been disregarded for the most part. This work presents an optimization of an existing peptic digest protocol for the analysis of membrane proteins using bacteriorhodopsin from purple membranes as reference.

The recently established coupling of a MALDI-type ion source to a linear ion trap and an orbitrap mass analyzer offers high-accuracy mass measurements compared to common MALDI-TOF/TOF instruments. Contrary to MALDI-TOF/TOF, the fragmentation of peptides in the new hybrid mass spectrometer is less efficient due to the generation of predominantly singly charged ions by the MALDI process. Therefore, data from two MALDI instruments, TOF/TOF and Orbitrap, were combined into a single data set in order to obtain accurate precursor masses as well as superior MS/MS spectra. This study demonstrates that an accurate precursor mass is particularly important for the nLC-MS/MS analyses of less-specific proteolytic digests. A potential gain of approximately one-third additional peptides identifications was theoretically estimated from previously published MALDI-TOF/TOF data. These calculations were verified by the nLC-MS/MS analysis of two elastatically digested proteomes, one cytosolic (Corynebacterium glutamicum) and one membrane (Halobacterium salinarium). Thereby it was discovered that the error distribution of a MALDI-Orbitrap can be significantly improved by applying an easy recalibration strategy. In summary, this study represents an updated workflow for the analysis of less-specific digests using nLC-MALDI.

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.

Progress in proteomics is mainly driven by advances in mass spectrometric (MS) technologies. Here we benchmarked the performance of the latest MS instrument in the benchtop Orbitrap series, the Q Exactive HF-X, against its predecessor for proteomics applications. A new peak-picking algorithm, a brighter ion source, and optimized ion transfers enable productive MS/MS acquisition above 40 Hz at 7500 resolution. The hardware and software improvements collectively resulted in improved peptide and protein identifications across all comparable conditions, with an increase of up to 50 percent at short LC-MS gradients, yielding identification rates of more than 1000 unique peptides per minute. Alternatively, the Q Exactive HF-X is capable of achieving the same proteome coverage as its predecessor in approximately half the gradient time or at 10-fold lower sample loads. The Q Exactive HF-X also enables rapid phosphoproteomics with routine analysis of more than 5000 phosphopeptides with short single-shot 15 min LC-MS/MS measurements, or 16 700 phosphopeptides quantified across ten conditions in six gradient hours using TMT10-plex and offline peptide fractionation. Finally, exciting perspectives for data-independent acquisition are highlighted with reproducible identification of 55 000 unique peptides covering 5900 proteins in half an hour of MS analysis.

In the yeast Saccharomyces cerevisiae as in other eukaryotes non-polar lipids are a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerols (TG) and steryl esters (SE) are stored in so-called lipid particles/droplets (LP) as biologically inert form of fatty acids and sterols. To understand LP structure and function in more detail we investigated the molecular equipment of this compartment making use of mass spectrometric analysis of lipids (TG, SE, phospholipids) and proteins. We addressed the question whether or not lipid and protein composition of LP influence each other and performed analyses of LP from cells grown on two different carbon sources, glucose and oleate. Growth of cells on oleate caused dramatic cellular changes including accumulation of TG at the expense of SE, enhanced the amount of glycerophospholipids and strongly increased the degree of unsaturation in all lipid classes. Most interestingly, oleate as a carbon source led to adaptation of the LP proteome resulting in the appearance of several novel LP proteins. Localization of these new LP proteins was confirmed by cell fractionation. Proteomes of LP variants from cells grown on glucose or oleate, respectively, were compared and are discussed with emphasis on the different groups of proteins detected through this analysis. In summary, we demonstrate flexibility of the yeast LP lipidome and proteome and the ability of LP to adapt to environmental changes., Highlights ► Molecular analysis of yeast lipid particles/droplets components. ► Combination of lipidome and proteome analysis. ► Effect of oleate stress on lipid particle lipidome and proteome. ► High flexibility of lipid particles on environmental conditions.