Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation.

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.

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion.

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016.

Characterization of post-translational modifications in full-length human BMP-1 confirms the presence of a rare vicinal disulfide linkage in the catalytic domain and highlights novel features of the EGF domain.

UNLABELLED: 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.

Characterization and function of the first antibiotic isolated from a vent organism: the extremophile metazoan Alvinella pompejana.

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.

Determination of disulfide linkages in antimicrobial peptides of the macin family by combination of top-down and bottom-up proteomics.

Macins are a distinct class of antimicrobial peptides (AMPs) produced by leeches and Hydra. Their function depends strongly on their three-dimensional structure. In order to support structural elucidation of these AMPs, the knowledge and proper assignment of disulfide bonds formed in these cysteine-rich peptides is a prerequisite. In this report, we outline an analytical strategy, encompassing a combination of top-down MS based analytics and sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution MS/MS analysis by LTQ-Orbitrap MS to assign disulfide linkages of three members of the macin family, namely neuromacin, theromacin, and hydramacin-1. The results revealed that the eight cysteine residues conserved in all three macins form the same four disulfide bonds, i.e. [C1:C6], [C2:C5], [C3:C7], and [C4:C8]. Theromacin, which possess two additional cysteine residues, forms a fifth disulfide bond. BIOLOGICAL SIGNIFICANCE: Beside the high biological significance which is based on the inherent dependence of biological activity on the structural features of antimicrobial peptides (which holds true for entirely every protein), the presented analytical strategy will be of wide interest, as it widens the available toolbox for the analysis of this important posttranslational modification.

Upd3--an ancestor of the four-helix bundle cytokines.

The unpaired-like protein 3 (Upd3) is one of the three cytokines of Drosophila melanogaster supposed to activate the JAK/STAT signaling pathway (Janus tyrosine kinases/signal transducer and activator of transcription). This activation occurs via the type-I cytokine receptor domeless, an orthologue of gp130, the common signal transducer of all four-helix bundle interleukin-6 (IL-6) type cytokines. Both receptors are known to exist as preformed dimers in the plasma membrane and initiate the acute-phase response. These facts indicate an evolutionary relation between vertebrate IL-6 and the Drosophila protein Upd3. Here we presented data which strengthen this notion. Upd3 was recombinantly expressed, a renaturation and purification protocol was established which allows to obtain high amounts of biological active protein. This protein is, like human IL-6, a monomeric-α helical cytokine, implicating that Upd3 is an "ancestor" of the four-helix bundle cytokines.

Optimized fragmentation conditions for iTRAQ-labeled phosphopeptides.

Protein phosphorylation is an important post-translational modification that plays a regulatory role within numerous biological processes. The simultaneous identification, localization, and quantification of phosphorylated proteins is vital for understanding this dynamic control mechanism. The application of isobaric labeling strategies, for example, iTRAQ, for quantitative phosphopeptide analysis requires simultaneous monitoring of peptide backbone fragmentation, loss of phosphoryl moieties, and the cleavage of isobaric labeling reporter ions. In the present study, we have examined MS/MS fragmentation modes available in the Orbitrap Velos MS (collision induced dissociation (CID), CID plus multistage activation, and higher energy collision dissociation (HCD)), for their ability to generate ions required for simultaneous quantification and identification of iTRAQ labeled phosphopeptides in a semicomplex (12) and a complex (131) phosphopeptide mix. The required normalized collision energies for quantification and identification of iTRAQ-labeled phosphopeptides require a compromise between the optimal parameters for each aspect. Here, we were able to determine an optimized MS/MS measurement protocol that involves CID measurement in ion trap for identification followed by HCD measurement for parallel identification and quantification that satisfies the time requirements for LC-MS/MS experiments.

Membrane-proximal domain of a disintegrin and metalloprotease-17 represents the putative molecular switch of its shedding activity operated by protein-disulfide isomerase.

A disintegrin and metalloprotease-17 (ADAM17) is a major sheddase responsible for the regulation of a wide range of biological processes, like cellular differentiation, regeneration, or cancer progression. Hitherto, the mechanism regulating the enzymatic activity of ADAM17 is poorly understood. Recently, protein-disulfide isomerase (PDI) was shown to interact with ADAM17 and to down-regulate its enzymatic activity. Here we demonstrate by NMR spectroscopy and tandem-mass spectrometry that PDI directly interacts with the membrane-proximal domain (MPD), a domain of ADAM17 involved in its dimerization and substrate recognition. PDI catalyzes an isomerization of disulfide bridges within the thioredoxin motif C600XXC603 of the MPD and results in a drastic structural change between an active open state and an inactive closed conformation. This conformational change of the MPD putatively acts as a molecular switch, facilitating a global reorientation of the extracellular domains in ADAM17 and regulating its shedding activity.

Nano-high-performance liquid chromatography with online precleaning coupled to inductively coupled plasma mass spectrometry for the analysis of lanthanide-labeled peptides in tryptic protein digests.

Low background signals are an indispensable prerequisite for accurate quantification in bioanalytics. This poses a special challenge when using derivatized samples, where excess reagent concentrations are increasing the background signal. Precleaning steps often are time-consuming and usually lead to analyte losses. In this study, a set of labeled model peptides and a protein digest was analyzed using inductively coupled plasma mass spectrometry (ICPMS), coupled to nano ion pairing reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC). In addition, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for peptide identification. Peptides were labeled with lanthanide metals using bifunctional DOTA-based (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) reagents. The resulting metal excess was removed online during nano-HPLC, by trapping the labeled peptides on a C18-precolumn and washing them prior to their elution to the analytical column. Different ion pairing reagents like TFA (trifluoroacetic acid) and HFBA (heptafluorobutyric acid) were used in the study to enhance interactions of the different peptide species with the C18 material of the precolumn. HFBA even allowed the detection of a highly hydrophilic peptide that was not retained using TFA. It was shown that for the mixture of labeled model peptides, even a short 3 min washing step already enhanced the removal of the excess reagents significantly, whereas peptide losses were observable starting with a 10 min washing time. A 6 min washing time was determined to be the best parameter for lowering the lanthanide metal background while maintaining maximum peptide recovery. Alternative precleaning setups using EDTA to enhance the removal of free metal or an offline approach using solid phase extraction did not show promising results. The application of the optimized method to labeled peptides in a lysozyme digest showed results comparable to those obtained with model peptides.

Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba.

Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporin's active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.

Enzyme-cleavable tandem peptides for quantitative studies in MS-based proteomics.

A novel type of peptide standard is introduced that consists of two peptides combined in one synthetic molecule and separated by a proteolytic cleavage site. Upon enzymatic digestion, the two peptides are released in a molar one-to-one ratio. This method enables the generation of exact equimolar mixtures of two peptides of any nature and origin, thereby providing a valuable tool for the investigation of fundamental phenomena in MS. The applicability of the method is exemplified by the analysis of the effect of peptide sequence variations on the relative ionization efficiency in ESI- and MALDI-MS.

Macin family of antimicrobial proteins combines antimicrobial and nerve repair activities.

The tertiary structures of theromacin and neuromacin confirmed the macin protein family as a self-contained family of antimicrobial proteins within the superfamily of scorpion toxin-like proteins. The macins, which also comprise hydramacin-1, are antimicrobially active against Gram-positive and Gram-negative bacteria. Despite high sequence identity, the three proteins showed distinct differences with respect to their biological activity. Neuromacin exhibited a significantly stronger capacity to permeabilize the cytoplasmic membrane of Bacillus megaterium than theromacin and hydramacin-1. Accordingly, it is the only macin that displays pore-forming activity and that was potently active against Staphylococcus aureus. Moreover, neuromacin and hydramacin-1 led to an aggregation of bacterial cells that was not observed with theromacin. Analysis of the molecular surface properties of macins allowed confirmation of the barnacle model as the mechanistic model for the aggregation effect. Besides being antimicrobially active, neuromacin and theromacin, in contrast to hydramacin-1, were able to enhance the repair of leech nerves ex vivo. Notably, all three macins enhanced the viability of murine neuroblastoma cells, extending their functional characteristics. As neuromacin appears to be both a functional and structural chimera of hydramacin-1 and theromacin, the putative structural correlate responsible for the nerve repair capacity in leech was located to a cluster of six amino acid residues using the sequence similarity of surface-exposed regions.

Tandem mass tag protein labeling for top-down identification and quantification.

Top-down mass spectrometry holds tremendous potential for characterization and quantification of intact proteins. So far, however, very few studies have combined top-down proteomics with protein quantification. In view of the success of isobaric mass tags in quantitative bottom-up proteomics, we applied the tandem mass tag (TMT) technology to label intact proteins and examined the feasibility to directly quantify TMT-labeled proteins. A top-down platform encompassing separation via ion-pair reversed-phase liquid chromatography using monolithic stationary phases coupled online to an LTQ-Orbitrap Velos electron-transfer dissociation (ETD) mass spectrometer (MS) was established to simultaneously identify and quantify TMT-labeled proteins. The TMT-labeled proteins were found to be readily dissociated under high-energy collision dissociation (HCD) activation. The liberated reporter ions delivered expected ratios over a wide dynamic range independent of the protein charge state. Furthermore, protein sequence tags generated either by low-energy HCD or ETD activation along with the intact protein mass information allow for confident identification of small proteins below 35 kDa. We conclude that the approach presented in this pilot study paves the way for further developments and numerous applications for straightforward, accurate, and multiplexed quantitative analysis in protein chemistry and proteomics.

Uncoupling of bait-protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP-1 and the PKA Cbeta1 subunit.

An expression-uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP-bait protein from the target cells. Here, the TAP-tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue-specific binding partners of the protein kinase A catalytic subunit Cbeta1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor-dependent interaction of Cbeta1 with the cell cycle and apoptosis regulatory protein-1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell- and tissue-specific protein complexes.

Phosphorylated human galectin-3: facile large-scale preparation of active lectin and detection of structural changes by CD spectroscopy.

Galectin-3 has a unique modular design. Its short N-terminal stretch can be phosphorylated, relevant for nuclear export and anti-anoikis/apoptosis activity. Enzymatic modification by casein kinase 1 at constant ATP concentration yielded mg quantities of mono- and diphosphorylated derivatives at Ser5/Ser11 in a 2:1 ratio. Their carbohydrate-inhibitable binding to asialofetuin, cell surfaces of three tumor lines, rabbit erythrocytes leading to haemagglutination and cytoplasmic sites in fixed tissue sections was not markedly altered relative to phosphate-free galectin-3. Spectroscopically, phosphorylation induced alterations in the far UV CD, indicative of an increase in ordered structure. This is accompanied by changes in the environment of aromatic amino acids signified by shifts in the near UV CD.

Collision-induced reporter fragmentations for identification of covalently modified peptides.

Collision-induced reporter fragmentations of the currently most important covalent peptide modifications as detected by tandem mass spectrometry are summarized. These fragmentations comprise the formation of reporter ions, which are preferentially immonium ions, immonium ion-derived fragments or side chain fragments. In addition, the reporter neutral loss reactions for covalently modified amino acid residues are summarized. For each individual covalent modification which can be recognized by a reporter fragmentation, the accurate mass shift and the gross formula shift of the modified amino acid residue are given. The same set of data is provided for the reporter fragmentations. Finally, an extensive accurate mass and gross formula list is presented as supplementary material, describing mostly regular and modified y(1) and dipeptide a and b ions, which are helpful for identification of the peptide ends of covalently modified peptides.

Neutral loss-based phosphopeptide recognition: a collection of caveats.

The standard strategy for analysis by tandem mass spectrometry of protein phosphorylation at serine or threonine utilizes the neutral loss of H3PO4 (= 97.977/z) from proteolytic peptide molecular ions as marker fragmentation. Manual control of automatically performed neutral loss-based phosphopeptide identifications is strongly recommended, since these data may contain false-positive results. These are connected to the experimental neutral loss m/z error, to competing peptide fragmentation pathways, to limitations in data interpretation software, and to the general growth of protein sequence databases. The fragmentation-related limitations of the neutral loss approach cover (i) the occurrence of abundant 'close-to-98/z' neutral loss fragmentations, (ii) the erroneous assignment of a neutral loss other than loss of H3PO4 due to charge state mix-up, and (iii) the accidental occurrence of any fragment ion in the m/z windows of interest in combination with a charge-state mix-up. The 'close-to-98/z' losses comprise loss of proline (97.053/z), valine (99.068/z), threonine (101.048/z), or cysteine (103.009/z) preferably from peptides with N-terminal sequences PP, VP, TP, or CP, and loss of 105.025/z from alkylated methionine. Confusion with other neutral losses may occur, when their m/z window coincides with a 98/z window as result of a charge state mix-up. Neutral loss of sulfenic acid from oxidized methionine originating from a doubly charged precursor (63.998/2 = 31.999) may thus mimic the loss of phosphoric acid from a triply charged phosphopeptide (97.977/3 = 32.659). As a consequence of the large complexity of proteomes, peptide sequence ions may occur in one of the mass windows of H3PO4 loss around 97.977/z. Practical examples for false-positive annotations of phosphopeptides are given for the first two groups of error. The majority of these can be readily recognized using the guidelines presented in this study.

pI-based phosphopeptide enrichment combined with nanoESI-MS.

IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit pI values, which overlap in the range of about 4-6. However, after methyl esterification of all carboxyl functions, the pI values of tryptic peptides and phosphopeptides regroup in discrete clusters. In addition, mono- and diphosphorylated peptides show different but very homogeneous pI values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3-10 as used in the first dimension of 2-DE. After IEF of methyl-esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI-MS. Phosphopeptides were found to focus in good agreement with their calculated pI values. This analytical strategy showed a resolution of about 0.2 pI units, and thus turned out to be capable of detecting minor differences in pI values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a pI range of 3-10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to pI is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics.

Effective solvation of alkaline earth ions by proline-rich proteolytic peptides of galectin-3 upon electrospray ionisation.

In an analysis of a combined chymotrypsin/AspN digest of galectin-3 by positive ion nano-electrospray ionisation mass spectrometry (nanoESI-MS) several peptides were observed which showed metal adduct ions as their most abundant ion signals. The most prominent adduct ions were observed at m/z values corresponding to [M+40]2+, [M+41]3+, and [M+42]4+ ions. Detailed investigation of the [M+40]2+ ion of the peptide GAPAGPLIVPY showed that it was not, as originally expected, a [M+H+39K]2+ adduct ion but had the composition [M+40Ca]2+. This was verified by several approaches: (i) nanoESI-MS/MS of the [M+Ca]2+ adduct ions resulted in the virtually exclusive formation of doubly charged fragment ions; (ii) mass determination by quadrupole time-of-flight (QTOF)-MS provided a preliminary identification; and (iii) accurate mass measurement using nanoESI Fourier transform ion cyclotron resonance (FTICR)-MS at a mass resolving power of 500 000 allowed the specific detection and identification of the isobaric ion pairs [M+40Ca]2+/[M+H+39K]2+ and [M+24Mg]2+/[M+H+23Na]2+. All peptides in the chymotryptic galectin-3 digest without a basic residue (K or R) showed addition of calcium as the most prominent ionisation principle. A further common feature of these nonbasic peptides was the presence of several proline residues, which is assumed to be a factor promoting the intense addition of calcium. It was observed that the common trace levels of sodium and calcium in analytical grade solvents (about 1-10 microM) are sufficient to generate the [M+H+23Na]2+ and [M+40Ca]2+ ions as the most prominent species of the peptide GAPAGPLIVPY. We conclude that the sequence motifs P-XX-P and P-XXX-P favour the solvation of alkaline earth ions in ESI-MS. In view of the successful detection of physiological Ca/protein interactions by ESI-MS, this finding may point to a solvation of Ca2+ by galectin in solution. The findings open new routes of research in the study of metal/protein and metal/peptide interactions

Iodoacetamide-alkylated methionine can mimic neutral loss of phosphoric acid from phosphopeptides as exemplified by nano-electrospray ionization quadrupole time-of-flight parent ion scanning.

Formation of S-carbamidomethylmethionine (camMet) occurs as a side reaction during cysteine alkylation with iodoacetamide (IAA). In collision-induced dissociation, peptides with camMet show an abundant neutral loss of 2-(methylthio)acetamide (C3H7NOS = 105.025 Da) at moderate collision offset values which are similar to those optimal for loss of phosphoric acid (H3PO4 = 97.977 Da). Neutral loss analysis is used for spotting of phosphopeptides which contain phosphoserine (pSer) or phosphothreonine (pThr) residues. In the case where precursor ions cannot be accurately assigned in the survey spectrum (e.g. due to low ion abundance or signal overlap), the mass accuracy of a neutral loss tandem mass spectrometry (MS/MS) analysis depends on the precursor ion isolation window. For the charge states 2+, 3+ or 4+, a typical 3.5 Da precursor isolation window results in neutral loss windows of 7, 10.5 or 14 Da, respectively. Consequently, neutral loss of 105 Da from alkylated methionine residues can mimic the phosphoserine/phosphothreonine-specific neutral loss of 98 Da. In the evaluation of quadrupole time-of-flight (QTOF) parent ion scan data for neutral loss of H3PO4, this interference was frequently observed. It is illustrated in this study using the analysis of ovalbumin phosphorylation as an example. The +80 Da molecular weight shift connected with phosphorylation at serine or threonine may also be mimicked by carbamidomethylation of methionine through a combination with sodium adduction (+57 Da +22 Da = +79 Da). For highly sensitive neutral loss analysis of serine and threonine phosphorylation, careful data inspection is recommended if reduction and alkylation by IAA is employed.