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Due to mechanisms such as proteolytic processing or alternative translation starts, in vivo proteoforms do not necessarily correspond directly to those encoded in the genome. Therefore, the knowledge of protein termini is an indispensable prerequisite to understand protein functions. So far, sequencing of protein N- and C-termini has been limited to single purified protein species, while the proteome-wide identification of N- and C-termini relies on the generation of single, terminal proteotypic peptides followed by chemical enrichment or depletion strategies to facilitate their detection via mass spectrometry (MS). To overcome the numerous limitations in such approaches, we present an alternative concept that readily enables unbiased ladder sequencing of protein N- and C-termini. The approach combines exopeptidase digestions of the proteome with two-dimensional chromatographic separation and tandem-MS. We demonstrate the potential of the methodology by analyzing the N- and C-terminome of

In top-down (TD) proteomics, prefractionation prior to mass spectrometric (MS) analysis is a crucial step for both the high confidence identification of proteoforms and increased proteome coverage. In addition to liquid-phase separations, gas-phase fractionation strategies such as field asymmetric ion mobility spectrometry (FAIMS) have been shown to be highly beneficial in TD proteomics. However, so far, only external compensation voltage (CV) stepping has been demonstrated for TD proteomics, i.e., single CVs were applied for each run. Here, we investigated the use of internal CV stepping (multiple CVs per acquisition) for single-shot TD analysis, which has huge advantages in terms of measurement time and the amount of sample required. In addition, MS parameters were optimized for the individual CVs since different CVs target certain mass ranges. For example, small proteoforms identified mainly with more negative CVs can be identified with lower resolution and number of microscans than larger proteins identified primarily via less negative CVs. We investigated the optimal combination and number of CVs for different gradient lengths and validated the optimized settings with the low-molecular-weight proteome of CaCo-2 cells obtained using a range of different sample preparation techniques. Compared to measurements without FAIMS, both the number of identified protein groups (+60-94%) and proteoforms (+46-127%) and their confidence were significantly increased, while the measurement time remained identical. In total, we identified 684 protein groups and 2675 proteoforms from CaCo-2 cells in less than 24 h using the optimized multi-CV method.

The proteome wide, mass spectrometry based identification of protein C-termini is hampered by factors such as poor ionization efficiencies, low yielding labeling strategies, or the need for enrichment procedures. We present a bottom-up proteomics workflow to identify protein C-termini utilizing a combination of strong cation exchange chromatography, on-solid phase charge-reversal derivatization and LC-MS/MS analysis. Charge-reversal improved both MS and MS/MS spectra quality of peptides carrying nonbasic C-terminal residues, allowing the identification of a high number of noncanonical C-termini not identified in nonderivatized samples. Further, we could show that C-terminal 18O labeling introduced during proteolytic processing of the samples is not suitable to distinguish internal from C-terminal peptides. The presented workflow enables the simultaneous identification of proteins by internal peptides and additionally provides data for the C- and N-terminome. Applying the developed workflow for the analysis of a Saccharomyces cerevisiae proteome allowed the identification of 734 protein C-termini in three independent biological replicates, and additional 789 candidate C-termini identified in two or one of three biological replicates, respectively. The developed analytical workflow allowed us to chart the nature of the yeast C-terminome in unprecedented depth and provides an alternative methodology to assess C-terminal proteolytic protein processing.

The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.

While LC-MS-based proteomics with high nanograms to micrograms of total protein has become routine, the analysis of samples derived from low cell numbers is challenged by factors such as sample losses, or difficulties encountered with the manual manipulation of small liquid volumes. Digital microfluidics (DMF) is an emerging technique for miniaturized and automated droplet manipulation, which has been proposed as a promising tool for proteomic sample preparation. However, proteome analysis of samples prepared on-chip by DMF has previously been unfeasible, due to incompatibility with down-stream LC-MS instrumentation. To overcome these limitations, we here developed protocols for bottom-up LC-MS based proteomics sample preparation of as little as 100 mammalian cells on a commercially available digital microfluidics device. To this end, we developed effective cell lysis conditions optimized for DMF, as well as detergent-buffer systems compatible with downstream proteolytic digestion on DMF chips and subsequent LC-MS analysis. A major step was the introduction of the single-pot, solid-phase-enhanced sample preparation (SP3) approach on-chip, which allowed the removal of salts and anti-fouling polymeric detergents, thus rendering sample preparation by DMF compatible with LC-MS-based proteome analysis. Application of DMF-SP3 to the proteome analysis of Jurkat T cells led to the identification of up to 2500 proteins from approximately 500 cells, and up to 1200 proteins from approximately 100 cells on an Orbitrap mass spectrometer, emphasizing the high compatibility of DMF-SP3 with low protein input and minute volumes handled by DMF. Taken together, we demonstrate the first sample preparation workflow for proteomics on a DMF chip device reported so far, allowing the sensitive analysis of limited biological material.

The identification and quantification of molecules involved in bacterial communication are major prerequisites for the understanding of interspecies interactions at the molecular level. We developed a procedure allowing the determination of 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and the virulence factor pyocyanin (PYO) formed by the Gram-negative bacterium Pseudomonas aeruginosa. The method is based on dispersive liquid-liquid microextraction from small supernatant volumes (below 10 μL) followed by quantitative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The use of ionic liquid matrix led to a lowered limit of detection for pyocyanin and, due to suppression of matrix background signals, easy to interpret mass spectra compared to crystalline matrices. Using an isotope-labeled pyocyanin standard synthesized in small-scale synthesis, quantitative analysis spanning approximately one order of magnitude (0.5 to 250 fmol) was feasible. The method was successfully applied to the detection of the signaling molecules PQS and HHQ in cultures of P. aeruginosa strains isolated from sputum of cystic fibrosis patients and allowed a highly sensitive quantification of PYO from these cultures. Hence, the developed method bears the potential to be used for screening purposes in clinical settings and will help to decipher the molecular basis of bacterial communication. Graphical abstract Ionic liquid matrices for the detection and quantification of the toxin pyocyanin and other signaling molecules from P. aeruginosa by MALDI MS.

Mass spectrometry based proteomics is an indispensable tool for almost all research areas relevant for the understanding of proteolytic processing, ranging from the identification of substrates, products and cleavage sites up to the analysis of structural features influencing protease activity. The majority of methods for these studies are based on bottom-up proteomics performing analysis at peptide level. As this approach is characterized by a number of pitfalls, e.g. loss of molecular information, there is an ongoing effort to establish top-down proteomics, performing separation and MS analysis both at intact protein level. We briefly introduce major approaches of bottom-up proteomics used in the field of protease research and highlight the shortcomings of these methods. We then discuss the present state-of-the-art of top-down proteomics. Together with the discussion of known challenges we show the potential of this approach and present a number of successful applications of top-down proteomics in protease research. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Analysis of the human skin proteome is key to understand molecular mechanisms maintaining health or leading to diseases of this important organ. For minimal invasive sampling of skin proteomes, the use of self-adhesive tape strips has been successfully applied. However, the methods previously presented were evaluated on different types of skin samples (e.g. healthy, diseased) and used a variety of cell lysis/protein extraction methods, which renders a systematic comparison and thus the identification of the most efficient protocols difficult. Here, we present a study comparing five different approaches for cell lysis and protein extraction from single tape strip biopsies. Extraction using a detergent mix or 1% SDS proved to be most efficient. Further, we replaced protein precipitation by single-pot, solid-phase-enhanced sample preparation (SP3), which strongly enhanced the number of identified proteins. This fully LC-MS compatible methodology provides a fast and reproducible approach for minimal invasive sampling of human skin proteomes. BIOLOGICAL SIGNIFICANCE: Fast and reproducible minimal invasive sampling of human skin proteomes is a major prerequisite for clinical proteomics studies aiming to decipher molecular mechanisms involved in the homeostasis as well as in the development of diseases. By optimization of tape strip sampling, e.g. the introduction of SP3 sample cleanup prior to LC-MS analysis, the presented protocol leads to yet not reported numbers of protein identifications from healthy human skin. Further, due to its efficiency it allows analysis from minimal sample amounts, e.g. from single tape strips, while established protocols relied on pooling of multiple tape strips. This provides the opportunity to perform spatially (lateral) resolved proteome analyses from different depths of the skin by analysis of consecutive strips.

International audience; Labeling of peptides and proteins using chelators binding metal ions has become a novel approach for quantitative proteomics in recent years. The aim of this work was the optimization of a new method for peptide derivatization with lanthanide labels (holmium, lutetium, and thulium) followed by nano high performance liquid chromatography (nano-HPLC) separation with UV detection. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI MS) was used to confirm the derivatization and to identify the derivatized peptides. Peptides were labeled with the three different lanthanide metals using a bifunctional DOTA-based (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) reagent. The results demonstrate that the derivatization reaction using the chelating reagent DOTA-NHS-ester was effective for single peptides and peptide mixtures. Furthermore, an efficient pre-cleaning method was applied by nano-HPLC using a C-18 trap column for elimination of the excess of labeling reagent. The application of the optimized method to label peptides in a Cytochrome C digest delivered comparable results to those obtained with model peptides. © 2014 Elsevier B.V.

N-acyl homoserine lactones (AHL) are small signal molecules involved in the quorum sensing of many gram-negative bacteria, and play an important role in biofilm formation and pathogenesis. Present analytical methods for identification and quantification of AHL require time-consuming sample preparation steps and are hampered by the lack of appropriate standards. By aiming at a fast and straightforward method for AHL analytics, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Suitable MALDI matrices, including crystalline and ionic liquid matrices, were tested and the fragmentation of different AHL in collision-induced dissociation MS/MS was studied, providing information about characteristic marker fragments ions. Employing small-scale synthesis protocols, we established a versatile and cost-efficient procedure for fast generation of isotope-labeled AHL standards, which can be used without extensive purification and yielded accurate standard curves. Quantitative analysis was possible in the low pico-molar range, with lower limits of quantification reaching from 1 to 5 pmol for different AHL. The developed methodology was successfully applied in a quantitative MALDI MS analysis of low-volume culture supernatants of Pseudomonas aeruginosa. Graphical abstract ᅟ.

A Disintegrin and Metalloproteinase 10 (ADAM10) and ADAM17 catalyze ectodomain shedding of a number of cell surface proteins important for embryonic development and tissue homeostasis. Changes in the expression levels or dysregulated proteolytic activity of ADAM10 and ADAM17 have been shown to play important roles in multiple diseases such as inflammation, cancer, and neurodegenerative disorders. Despite the well documented substrate repertoire of ADAM10 and ADAM17, little is known about their cleavage site specificity. We optimized Q-PICS (Quantitative Proteomics for the Identification of Cleavage Sites) to elucidate the cleavage site specificity of recombinant murine ADAM10 and ADAM17. Two different yeast proteome-derived peptide libraries were used and samples were analyzed by LC-MALDI and LC-ESI MS in parallel. We show that the largest difference in the cleavage site specificities of ADAM10 and ADAM17 is at the P1' site: while both enzymes cleave N-terminal of leucine, only ADAM10 shows additional preference toward aromatic amino acids, whereas ADAM17 exhibits the highest preference for valine. Together with further amino acid preferences more adjacent to the scissile bond, our data is in good agreement with ADAM10/17 cleavage sites previously identified in native substrates. Overall, the precise identification of ADAM10 and ADAM17 cleavage site specificity provides the basis for better substrate identification in vivo and the generation of specific inhibitors or activity based probes.

Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography–mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation., Author Summary Interleukin-6 (IL-6) is a cytokine secreted by our body upon infection or trauma to stimulate the immune system response. IL-6 is partially responsible for fever and triggers inflammation in many diseases. It activates its target cells via the membrane-bound IL-6 receptor (IL-6R), and soluble forms of this receptor (sIL-6R) are present in high amounts in the serum of healthy individuals and mediate the inflammatory response in all cells of the human body. However, it remains unclear how the soluble form of this cytokine is generated in humans. In this study, we isolate sIL-6R from human serum and show that the majority is produced via cleavage of the membrane-bound IL-6R by a protease. We identify the exact cleavage site and find that it is identical to a cleavage site used by the metalloprotease ADAM17. We further show that glycosylation, a post-transcriptional modification, is dispensable for the transport and biological function of IL-6R and map the occupancy of all O- and N-glycosylation sites. However, we find that only a single N-glycan is critically involved in the regulation of proteolysis by ADAM17 and conclude that glycosylation is an important regulator for sIL-6R generation.

Two peptide quantification strategies, the isobaric tags for relative or absolute quantitation (iTRAQ) labeling methodology and a metal-chelate labeling approach, were compared using matrix-assisted laser desorption/ionization-TOF/TOF MS and MS/MS analysis. Amino- and cysteine-directed labeling using the rare earth metal chelator 1,4,7,10-tetraazacyclododecane-N,N',N″,N″'-tetraacetic acid (DOTA) were applied for relative quantification of single peptides and a six-protein mixture. For analyte ratios close to one, iTRAQ and amino-directed DOTA labeling delivered overall comparable results regarding accuracy and reproducibility. In contrast, the MS-based quantification via amino-directed lanthanide-DOTA tags was more accurate for analyte ratios ≥5 and offered an extended dynamic range of three orders of magnitude. Our results show that the amino-directed DOTA labeling is an alternative relative quantification tool offering advantages like flexible multiplexing possibilities and, in particular, large dynamic ranges, which should be useful in sophisticated, targeted issues, where the accurate determination of extremely different protein or peptide concentration becomes relevant.

The recent discovery of an increasing number of small open reading frames (sORF) creates the need for suitable analytical technologies for the comprehensive identification of the corresponding gene products. For biological and functional studies the knowledge of the entire set of proteins and sORF gene products is essential. Consequently in the present study we evaluated analytical approaches that will allow for simultaneous analysis of widest parts of the proteome together with the predicted sORF. We performed a full proteome analysis of the methane producing archaeon Methanosarcina mazei strain Gö1 cytosolic proteome using a high/low pH reversed phase LC-MS bottom-up approach. The second analytical approach was based on semi-top-down strategy, encompassing a separation at intact protein level using a GelFree system, followed by digestion and LC-MS analysis. A high overlap in identified proteins was found for both approaches yielding the most comprehensive coverage of the cytosolic proteome of this organism achieved so far. The application of the second approach in combination with an adjustment of the search criteria for database searches further led to a significant increase of sORF peptide identifications, finally allowing to detect and identify 28 sORF gene products.

Protease-catalyzed hydrolysis of peptide bonds is one of the most pivotal post-translational modifications fulfilling manifold functions in the regulation of cellular processes. Therefore, dysregulation of proteolytic reactions plays a central role in many pathophysiological events. For this reason, understanding the molecular mechanisms in proteolytic reactions, in particular the knowledge of proteases involved in complex processes, expression levels and activity of protease and knowledge of the targeted substrates are an indispensable prerequisite for targeted drug development. The present review focuses on mass spectrometry-based proteomic methods for the analysis of protease cleavage sites, including the identification of the hydrolyzed bonds as well as of the surrounding sequence. Peptide- and protein-centric approaches and bioinformatic tools for experimental data interpretation will be presented and the major advantages and drawbacks of the different approaches will be addressed. The recent applications of these approaches for the analysis of biological function of different protease classes and potential future directions will be discussed.

A brief survey is given of the last 2 years' literature on electrospray mass spectrometry (ESI-MS) for speciation analysis. As observed for many years, the main recent applications in this field concern arsenic and selenium species, especially in studies encompassing combined use of molecular and element mass spectrometry. A further application field is the stoichiometric characterization of metal complexes by ESI-MS, which in some studies was assisted by nuclear magnetic resonance spectroscopy. A few examples are presented to illustrate arsenic species involved in metabolic pathways, sulfur species in oils and bitumen, and aluminum complexes. On the basis of this review, we also give an outlook of expected future developments and trends in this research field.

Metal labelling of peptides and proteins using high-affinity metal-chelating compounds has found widespread applications in the medical and bioanalytical fields. In the present study we investigated the analysis of peptides derivatized either with cysteine- or amino group-directed metal-bound DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The metal complexes of DOTA were shown to be stable under MALDI-MS conditions. The introduction of the metal label led in a number of cases to significantly increased signal-to-noise (S/N) values and thus improved sensitivity of the labelled peptides compared to their unlabelled counterparts, especially for multiply labelled peptides. The presence of the labels did alter the tandem mass spectrometric (MS/MS) behaviour, namely the formation of sequence specific a-, b- and y-ion series, in dependence of the position of the label within the peptide sequence. For cysteine-derivatized peptides several label-specific reporter ions and characteristic immonium ions could be identified. Amino-directed labelling led only to the formation of characteristic immonium ions in e-amino groups of lysine, whereas N-terminal labelling in some cases led to the formation of a1- and b1-ions. The results clearly show that MALDI-MS is suitable for the analysis of metal-labelled peptides, which was also confirmed in liquid chromatography (LC)/MALDI-based identification of proteins in a model protein mixture labelled with Cys-reactive DOTA. Here, in comparison to a run with alkylated cysteines, more than 50% more cysteine-containing peptides were identified. Copyright © 2010 John Wiley & Sons, Ltd.

Method development for the analytics of biomolecules has an enormous impact on biochemistry, biology, medicine and life sciences. In particular, methods for peptide and protein quantification are required for the study of the proteome of organisms in order to understand fundamental processes of life. Most popular approaches use stable isotope labeling and molecular mass spectrometry (MS). This review highlights recent quantification strategies based on biomolecule labeling with metal tags and inductively-coupled plasma MS (ICP-MS) and their parallel characterization by molecular MS. This new discipline at the interface of analytical chemistry and biochemistry is a rapidly growing research field. It includes the chemical derivatization of functional groups in proteins (e.g., with lanthanide chelates, ferrocenes, organomercury compounds and iodine), and their ultra-sensitive detection and quantification by ICP-MS, so that limits of detection for proteins down to the attomole level have been achieved. Quantification is realized with simple metal standards or stable metal isotopes (isotope-dilution analysis). We critically discuss different approaches and emphasize the prospects of future needs and developments.

Relative protein abundances of Escherichia coli MG1655 growing exponentially on minimal medium with acetate or glucose as the sole carbon source were investigated in a quantitative shotgun proteome analysis with TMT6-plex isobaric tags. Peptides were separated by high resolution high/low pH 2D-LC, using an optimized fraction pooling scheme followed by mass spectrometric analysis. Quantitative data were acquired for 2099 proteins covering 49% of the predicted E. coli proteins, showing system-wide effects of growth conditions. In total, 507 proteins showed a fold change of at least 1.5 and 205 proteins changed by more than twofold. Significant differences in abundance were observed for most of the proteins in the central carbon metabolism and in proteins relevant for amino acid and protein synthesis, processing of environmental information and scavenging of a variety of alternate carbon sources. Periplasmic-binding proteins were also more abundant on acetate, especially proteins involved in scavenging extracellular resources such as sugars. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD003863).

Since the introduction of ionic liquid matrices (ILM) for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, manifold applications for a range of different substance classes (e.g., amino acids, peptides or proteins) were described. ILM are composed of an equimolar mixture of classically used acidic MALDI matrices and organic bases, which allows for an almost infinite number of combinations with different properties. A major advantage offered by many ILM compared to classical crystalline matrices is a highly homogeneous sample distribution, which is especially important in regard to quantification.

Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications. Ten disulfide linkages of BMP-1, including the vicinal disulfide linkage C185-C186 could be unambiguously identified. Further, around 50% of this vicinal disulfide bond was found to be modified by N-ethylmaleimide (NEM), a cysteine protease inhibitor supplied when the BMP-1-containing medium was collected, suggesting that this bond was highly unstable. In the absence of NEM, BMP-1 has a higher tendency to form aggregates, but after aggregate removal, C185 and C186 are almost quantitatively engaged in the vicinal disulfide bond and BMP-1 activity remains unchanged. In addition, three consensus N-glycosylation sites at N142, N363, and N599 could be identified together with a previously unknown O-glycosylation site and an Asn-hydroxylation. Significance An in-depth characterization of post-translational modifications of the full-length human BMP-1 produced in mammalian cells by MS was performed. A rare vicinal disulfide bond in the catalytic domain could be confirmed for the first time by mass spectrometry along with nine other proposed disulfide linkages of mature BMP-1. This vicinal disulfide bond can transiently open to form covalent adducts with the cysteine protease inhibitor (NEM) supplied in cell medium during protein harvesting. Further, we report a previously unknown O-glycosylation site and Asn–hydroxylation site, indicating a novel feature of BMP-1 in the EGF domain. The study clearly outlines the benefit of in-depth characterization of overexpressed proteins to deduce important protein modifications.

The production and consumption of mare’s milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows’ milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage.

Metallo-beta-lactamases (MBLs) are targets for medicinal chemistry as they mediate bacterial resistance to beta-lactam antibiotics. Electrospray-ionization mass spectrometry (ESI-MS) was used to study the inhibition by a set of mercaptocarboxylates of two representative MBLs with different optimal metal stoichiometries for catalysis. BcII is a dizinc MBL (Class B1), whilst the CphA MBL (Class B2) exhibits highest activity with a single zinc ion in the active site. Experimental parameters for the detection of the metallo-enzyme and the metallo-enzyme-inhibitor complexes were evaluated and optimized. Following investigations on the stoichiometry of metal binding, the affinity of the inhibitors was investigated by measuring the relative abundance of the complex compared to the metalloprotein. The results for the BcII enzyme were in general agreement with solution assays and demonstrated that the inhibitors bind to the dizinc form of the BcII enzyme. The results for the CphA(ZnII) complex unexpectedly revealed an increased affinity for the binding of a second metal ion in the presence of thiomandelic acid. The results demonstrate that direct ESI-MS analysis of enzyme:inhibitor complexes is a viable method for screening inhibitors and for the rapid assay of the enzyme:metal:inhibitor ratios.

Ionic liquid matrixes (ILM) have been shown to allow very homogeneous sample preparations, facilitating relative quantifications using internal standards in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In the present work, the ability to perform quantifications of peptides without using internal standards in these matrixes was investigated. Linear correlations between peptide amount and signal intensities could be observed when increased molar matrix-to-analyte ratios were applied. The dynamic range of linearity was approximately 1 order of magnitude. The method was applied successfully to monitor the time-dependent evolution of substrates and products in trypsin-catalyzed digests of single peptides and peptide mixtures. Thus, ionic liquid matrixes allow quantitative MALDI-MS without the need for internal standards, making the method a suitable tool for the fast screening of new enzymes or the search for substrates or inhibitors.

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a beta-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.

Ionic liquids are interesting solvents for a number of applications in chemistry and biotechnology. We characterized five different ionic liquids by laser desorption/ionization (LDI) and by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and studied the analysis of amino acids, peptides and proteins dissolved in these solvents. Signals of both anions and cations of the ionic liquids could be observed both in LDI- and in MALDI-MS. In the latter case, adduct formation between anions and cations of the analytes was observed. Amino acids, peptides and proteins could be analyzed in ionic liquids after addition of matrix substances. Sodium and potassium adducts were not observed in any analysis involving ionic liquids. Low molecular mass compounds and peptides could be analyzed best in the presence of water-immiscible ionic liquids, whereas proteins gave the best results in water-miscible ionic liquids. Optimal analysis conditions such as molar matrix-to-analyte and ionic liquid-to-matrix ratios were determined. Homogeneity of samples in the presence of ionic liquids was reduced compared with classical MALDI preparations. Relative quantitation of amino acids was possible using isotope-labeled internal standards. MALDI-MS thus can be used for the analysis of chemical reactions and the screening of enzyme-catalyzed reactions in ionic liquids and for the analysis of the biocatalysts dissolved in these solvents. Theoretical aspects of ion formation in the presence of ionic liquids both in LDI and MALDI analysis are discussed.

Quantitative matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) was applied for the screening of ten pyranose oxidase variants. Quantitative MALDI-ToF MS using isotopic labeled internal standards and ionic liquid matrices was performed using aliquots of enzyme reaction mixtures without prepurification steps. The results obtained were in good agreement with HPLC measurements. Analysis time was approx. 3.5 min for a five-fold determination. Thus, quantitative MALDI-ToF MS can be used as a tool for screening of sugar converting enzymes.

A major problem hampering the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for quantitative measurements is the inhomogeneous distribution of analytes and matrices in solid sample preparations. The use of ionic liquids as matrices for the qualitative and quantitative analysis of low molecular weight compounds like amino acids, sugars and vitamins was investigated. The ionic liquid matrices are composed of equimolar combinations of classical MALDI matrices (sinapinic acid, alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) with organic bases. These matrix systems allow a homogenous sample preparation with a thin ionic liquid layer having negligible vapour pressure. This leads to a facilitated qualitative and quantitative measurement of the analytes compared with classical solid matrices.

Oxidation of methionine residues in peptides and proteins occurs in vivo or may be an artifact resulting from purification steps. We present a three step method for the localization of methionine sulfoxides in peptides with two methionine residues. In the first step, the N-terminus as well as other reactive side chain functions are blocked by acetylation. The resulting protected peptides are cleaved by cyanogen bromide. The cleavage does not occur at methionine sulfoxide but only at reduced methionine residues forming new amino termini. The newly formed amino group is then derivatized with a bromine containing compound in the last step of the procedure. The resulting peptide can easily be identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using both the characteristic isotope pattern of the halogen and the metastable loss of methanesulfenic acid from oxidized residues. This procedure allows the unequivocal localization of oxidized methionines even in complex peptide mixtures.

Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study, we used matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) to investigate amyloid deposits in formalin-fixed and paraffin-embedded tissue samples. Utilizing a novel peptide filter method, we found a universal peptide signature for amyloidoses. Furthermore, differences in the peptide composition of ALλ and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI-IMS MSI analysis, we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample-consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's protein composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis.

Sample preparation methods and data acquisition protocols were optimized for the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to high-throughput quantitative analysis of low molecular mass substrates and products of an enzyme-catalyzed reaction. Using a deuterlum-labeled internal standard, precise standard curves were obtained (r(2) = 0.9998) over two orders of magnitude of concentration of rac-1-phenylethylamine (PEA), which is converted to 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) by a lipase-catalyzed reaction with ethylmethoxyacetate (EMA) as second substrate. Reliable relative standard deviations were achieved (< or =5%) using automated analysis with peak intensity ratios between 0.2 and 5 of analyte to internal standard. This method permitted quantitative analysis of the lipase reaction, producing results comparable to those from gas chromatographic (GC) analysis in the dynamic range of GC. This work shows that MALDI-TOFMS can be applied for the high-throughput screening of enzymes.

Reversible binding of small compounds through hydrophobic interactions or hydrogen bonding to food proteins (e.g. milk proteins) is a thoroughly researched topic. In contrast, covalent interactions are not well characterized. Here, we report a rare form of positive-cooperativity-linear binding of allyl isothiocyanate with β-lactoglobulin, resulting in the cleavage of a disulfide bond of the protein. We compared three methods (i.e. fluorescence quenching, equilibrium dialysis, and headspace-water equilibrium) to characterize the binding kinetics and investigated the molecular binding by mass spectrometry. The methodologies used were found to be comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium-based methods respectively.

Phosphopeptides are a useful tool for the investigation of phosphorylation as a reversible post-translational modification. There is a growing interest in using mimics of phosphoamino acids involved in phosphorylation in order to study the enzymes concerned in these processes. These mimics should contain a non-hydrolysable or isoelectrically modified phosphate moiety to be used as a specific inhibitor of phosphatases and kinases. We introduce solid-phase synthesis of H- and methylphosphonopeptides as a new class of mimics of phosphotyrosyl peptides. The peptides were synthesized on solid phase using the standard fluorenyl-methyloxycarbonyl (Fmoc) strategy. Tyrosine residues were incorporated as allyl-protected derivatives, which were selectively deprotected on the resin by treatment with Pd(PPh3)4. The peptide resin carrying the side-chain unprotected tyrosine of the model peptide Gly-Gly-Tyr-Ala was phosphonylated with di-tert-butyl-N,N-diethyl-phosphoramidite in the presence of 1H-tetrazole, yielding H-phosphonopeptides after trifluoroacetic acid (TFA) cleavage. Alternatively, phosphonylation of the unprotected tyrosine with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite catalysed by 1H-tetrazole and followed by oxidation led to the methylphosphonopeptides after TFA cleavage. We obtained both the H-phosphonopeptides and the methylphosphonopeptides of the tetrapeptide in high yields and purities above 90%, according to reversed-phase high-performance liquid chromatography (RP-HPLC). To investigate the general applicability of our new methodology, we synthesized phosphonopeptides up to 13 amino acids long, corresponding to recognition sequences of tyrosine kinases. After cleavage and deprotection, all phosphonopeptides were obtained in high yields and purities of about 90%, as shown by mass spectrometry. The only by-product found was the unmodified peptide. © 1997 European Peptide Society and John Wiley & Sons, Ltd.

Background The robust identification of isotope patterns originating from peptides being analyzed through mass spectrometry (MS) is often significantly hampered by noise artifacts and the interference of overlapping patterns arising e.g. from post-translational modifications. As the classification of the recorded data points into either ‘noise’ or ‘signal’ lies at the very root of essentially every proteomic application, the quality of the automated processing of mass spectra can significantly influence the way the data might be interpreted within a given biological context. Results We propose non-negative least squares/non-negative least absolute deviation regression to fit a raw spectrum by templates imitating isotope patterns. In a carefully designed validation scheme, we show that the method exhibits excellent performance in pattern picking. It is demonstrated that the method is able to disentangle complicated overlaps of patterns. Conclusions We find that regularization is not necessary to prevent overfitting and that thresholding is an effective and user-friendly way to perform feature selection. The proposed method avoids problems inherent in regularization-based approaches, comes with a set of well-interpretable parameters whose default configuration is shown to generalize well without the need for fine-tuning, and is applicable to spectra of different platforms. The R package IPPD implements the method and is available from the Bioconductor platform (http://bioconductor.fhcrc.org/help/bioc-views/devel/bioc/html/IPPD.html).

A simple and fast screening method for the selection of fractions of first dimension separation to be analyzed in second dimension-MS/MS experiments in offline multidimensional liquid chromatographic separation schemes for shotgun proteome analysis was developed. The method is based on the measurement of total peptide content of the first dimension fractions by MALDI MS and was established using a tryptic digest of a bacterial proteome. The results of the screening process were in good agreement with those obtained in a detailed proteome analysis performed by RPxion-pair RP-MALDI TOF/TOF MS analysis. The method supports a straightforward planning of experiments, also enabling a reduction of overall measurement time in shotgun proteome analysis.

An extensive data set comprising 2660 unique protein identifications was obtained for the proteome of a human brain tumor (glioblastoma multiforme) by combining the results of two complementary analytical strategies based on two-dimensional chromatography and mass spectrometry. A bottom-up method, performing peptide separation in both chromatographic dimensions was employed as well as a semi-top-down method, in which intact proteins were separated in the first and tryptic peptides in the second dimension. The identified proteins were assigned to their molecular functions and compared to previously identified proteins of glioblastoma multiforme (= astrocytoma WHO grade IV), lower WHO grade astrocytomas (grade II and III), and nontumor brain tissue. With the use of a subset of 104 identified membrane proteins, the properties of intact protein fractionation in the first dimension of the semi-top-down approach were elucidated in detail. The benefit of the semi-top-down approach was further demonstrated by the identification of a set of endogenous glioblastoma multiforme expressed proteins. These proteins correspond to recombinant antigens which were recently found to be reactive against autoantibodies in glioblastoma multiforme patients. The results indicate the usefulness of the semi-top-down approach for the investigation of immunogenic antigens in human tumor tissue samples.

Motivation: Mass spectrometry (MS) is one of the most important techniques for high-throughput analysis in proteomics research. Due to the large number of different proteins and their post-translationally modified variants, the amount of data generated by a single wet-lab MS experiment can easily exceed several gigabytes. Hence, the time necessary to analyze and interpret the measured data is often significantly larger than the time spent on sample preparation and the wet-lab experiment itself. Since the automated analysis of this data is hampered by noise and baseline artifacts, more sophisticated computational techniques are required to handle the recorded mass spectra. Obviously, there is a clear tradeoff between performance and quality of the analysis, which is currently one of the most challenging problems in computational proteomics. Results: Using modern graphics processing units (GPUs), we implemented a feature finding algorithm based on a hand-tailored adaptive wavelet transform that drastically reduces the computation time. A further speedup can be achieved exploiting the multi-core architecture of current computing devices, which leads to up to an approximately 200-fold speedup in our computational experiments. In addition, we will demonstrate that several approximations necessary on the CPU to keep run times bearable, become obsolete on the GPU, yielding not only faster, but also improved results. Availability: An open source implementation of the CUDA-based algorithm is available via the software framework OpenMS (http://www.openms.de). Contact: rene@bioinf.uni-sb.de; anhi@bioinf.uni-sb.de Supplementary information: Supplementary data are available at Bioinformatics online.

The repeatability of peptide identifications in shotgun proteome analyses employing strong cation-exchange-xion-pair RP HPLC hyphenated to ESI MS/MS was compared to an alternative scheme, comprising high-pH RP chromatography combined with low-pH ion-pair RP chromatography. Equivalent results were obtained with both methods in proteome analysis of Corynebacterium glutamicum. From a total number of 1350 to 1850 peptides identified in triplicate analyses of five consecutive fractions chosen from the first-dimension separation, 41-45% of the peptides were identified three times, whereas 16-22 and 37-39% of the peptides were identified only twice or once, respectively. A comparison of the repeatability of peptide identifications from complex samples upon 1- or 2-D chromatographic separation revealed that an additional separation dimension decreases the repeatability by approximately 25%.

The separation of complex peptide mixtures in shotgun proteome analysis using a 2D separation scheme encompassing reversed-phase × ion-pair reversed-phase (IP-RP) liquid chromatography coupled online to electrospray ion trap mass spectrometry (MS) has been shown earlier to be superior in terms of separation efficiency and technical robustness compared to the classically used separation scheme encompassing strong cation exchange × IP-RP-chromatography in shotgun proteome analysis. In the present study, this novel separation scheme was coupled offline to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF-MS for the analysis of the same sample, a tryptic digest of the cytosolic proteome of the bacterium Corynebacterium glutamicum. Compared to the earlier study, the MALDI-based platform led to a significantly increased number of peptides (7,416 vs. 2,709) and proteins (1,208 vs. 468, without single peptide-based identifications), respectively. This represents the majority of all predicted cytosolic proteins in C. glutamicum. The high proteome coverage, as well as the large number of low-abundant proteins identified with this improved analytical platform, pave the way for new biological studies.

In this work we present a novel approach for in vitro studies of cisplatin interactions with 8-mer oligonucleotides. The approach is based on the recently developed coupling of continuous elution gel electrophoresis (GE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) with the aim of monitoring the interaction process between this cytostatic drug and the nucleotides. In contrast to existing methods, the electrophoretic separation conditions used here allow both the determination of the reaction kinetics in more detail as well as the observation of dominant intermediates. Two different nucleotides sequences have been investigated for comparison purposes, one containing two adjacent guanines (5'-TCCGGTCC-3') and one with a combination of thymine and guanine (5'-TCCTGTCC-3'), respectively. In order to gain further structural information, MALDI-TOF MS measurements have been performed after fraction collection. This allows for identification of the intermediates and the final products and confirms the stepwise coordination of cisplatin via monoadduct to bisadduct formation. Furthermore, the ICP-MS results were quantitatively evaluated in order to calculate the kinetics of the entire process.

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.

Mass spectrometry (MS) has become today's de‐facto standard for high‐throughput analysis in proteomics research. Its applications range from toxicity analysis to MS‐based diagnostics. Often, the time spent on the MS experiment itself is significantly less than the time necessary to interpret the measured signals, since the amount of data can easily exceed several gigabytes. In addition, automated analysis is hampered by baseline artifacts, chemical as well as electrical noise, and an irregular spacing of data points. Thus, filtering techniques originating from signal and image analysis are commonly employed to address these problems. Unfortunately, smoothing, base‐line reduction, and in particular a resampling of data points can affect important characteristics of the experimental signal. To overcome these problems, we propose a new family of wavelet functions based on the isotope wavelet, which is hand‐tailored for the analysis of mass spectrometry data. The resulting technique is theoretically well‐foun...

A large number of matrix substances have been used for various applications in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The majority of matrices applied in ultraviolet-MALDI MS are crystalline, low molecular weight compounds. A problem encountered with many of these matrices is the formation of hot spots, which lead to inhomogeneous samples, thus leading to increased measurement times and hampering the application of MALDI MS for quantitative purposes. Recently, ionic (liquid) matrices (ILM or IM) have been introduced as a potential alternative to the classical crystalline matrices. ILM are equimolar mixtures of conventional MALDI matrix compounds such as 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxycinnamic acid (CCA) or sinapinic acid (SA) together with organic bases [e.g., pyridine (Py), tributylamine (TBA) or N,N-dimethylethylenediamine (DMED)]. The present article presents a first overview of this new class of matrices. Characteristic properties of ILM, their influence on mass spectrometric parameters such as sensitivity, resolution and adduct formation and their application in the fields of proteome analysis, the measurement of low molecular weight compounds, the use of MALDI MS for quantitative purposes and in MALDI imaging will be presented. Scopes and limitations for the application of ILM are discussed.

Matrix-assisted laser desorption/ionization mass spectrometry has become an indispensable tool for identification of proteins by peptide mass-fingerprint analysis. Selection of the matrix, addition of matrix additives, and sample-preparation techniques are known to affect the quality of the spectra and hence protein identification. We investigated the effect of pyridine as matrix additive for the commonly used crystalline matrix alpha-cyano-4-hydroxycinnamic acid (CCA), forming a pyridinium based ionic liquid matrix, on the mass spectra of synthetic peptides and tryptic protein digests. Beside the equimolar mixture of CCA and pyridine, the effect of addition of substoichiometric amounts of the base to the acid was tested. Optimum results in terms of signal-to-noise ratios, reduction of chemical noise, and reduced formation of alkali adducts and matrix clusters were observed for the matrix CCA-pyridine in the molar ratio 2:1. The optimized ionic liquid matrix was used for identification of tryptic digests of six model proteins and for identification of a protein extracted from a two-dimensional gel with the proteome of the bacterium Corynebacterium glutamicum, and shown to facilitate protein identification, yielding higher scores and increased sequence coverage compared with pure CCA. Thus CCA-Py 2:1 is a potential alternative for identification and characterization of proteins by peptide mass-fingerprint analysis.

Quantitative matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) was applied for the determination of concentrations of low-molecular-weight (

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a powerful tool for the measurement of low molecular mass compounds of biological interest. The limitations for this method are the volatility of many analytes, possible interference with matrix signals or bad ionization or desorption behavior of the compounds. We investigated the application of well-known and straightforward one-pot derivatization procedures to circumvent these problems. The derivatizations tested allow the measurement and the labeling of alcohols, aldehydes and ketones, carboxylic acids, alpha-ketocarboxylic acids and amines.