The One Hour Human Proteome.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.

Paradigm shift in biomarker translation: a pipeline to generate clinical grade biomarker candidates from DIA-MS discovery.

Clinical biomarker development has been stymied by inaccurate protein quantification from mass spectrometry (MS) discovery data and a prolonged validation process. To mitigate these issues, we created the Targeted Extraction Assessment of Quantification (TEAQ) software package. This innovative tool uses the discovery cohort analysis to select precursors, peptides, and proteins that adhere to established targeted assay criteria. TEAQ was applied to Data-Independent Acquisition MS data from plasma samples acquired on an Orbitrap™ Astral™ MS. Identified precursors were evaluated for linearity, specificity, repeatability, reproducibility, and intra-protein correlation from 11-point loading curves under three throughputs, to develop a resource for clinical-grade targeted assays. From a clinical cohort of individuals with inflammatory bowel disease (n=492), TEAQ successfully identified 1116 signature peptides for 327 quantifiable proteins from 1180 identified proteins. Embedding stringent selection criteria adaptable to targeted assay development into the analysis of discovery data will streamline the transition to validation and clinical studies.

Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition.

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th isolation windows, dissolving the differences between data-dependent and -independent methods. This is achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.

Fast and Deep Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer.

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence and structural context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data-Independent Acquisition.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.

Protein Coronas on Functionalized Nanoparticles Enable Quantitative and Precise Large-Scale Deep Plasma Proteomics.

Background: The wide dynamic range of circulating proteins coupled with the diversity of proteoforms present in plasma has historically impeded comprehensive and quantitative characterization of the plasma proteome at scale. Automated nanoparticle (NP) protein corona-based proteomics workflows can efficiently compress the dynamic range of protein abundances into a mass spectrometry (MS)-accessible detection range. This enhances the depth and scalability of quantitative MS-based methods, which can elucidate the molecular mechanisms of biological processes, discover new protein biomarkers, and improve comprehensiveness of MS-based diagnostics. Methods: Investigating multi-species spike-in experiments and a cohort, we investigated fold-change accuracy, linearity, precision, and statistical power for the using the Proteograph™ Product Suite, a deep plasma proteomics workflow, in conjunction with multiple MS instruments. Results: We show that NP-based workflows enable accurate identification (false discovery rate of 1%) of more than 6,000 proteins from plasma (Orbitrap Astral) and, compared to a gold standard neat plasma workflow that is limited to the detection of hundreds of plasma proteins, facilitate quantification of more proteins with accurate fold-changes, high linearity, and precision. Furthermore, we demonstrate high statistical power for the discovery of biomarkers in small- and large-scale cohorts. Conclusions: The automated NP workflow enables high-throughput, deep, and quantitative plasma proteomics investigation with sufficient power to discover new biomarker signatures with a peptide level resolution.

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data Independent Acquisition.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific™ Orbitrap™ mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

Online-2D NanoLC-MS for Crude Serum Proteome Profiling: Assessing Sample Preparation Impact on Proteome Composition.

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.

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients.

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.

Fast and Accurate Bacterial Species Identification in Urine Specimens Using LC-MS/MS Mass Spectrometry and Machine Learning.

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.

Performance Evaluation of the Q Exactive HF-X for Shotgun Proteomics.

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.

APP Is a Context-Sensitive Regulator of the Hippocampal Presynaptic Active Zone.

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 Aß-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.

A Mitochondrial Role of SV2a Protein in Aging and Alzheimer's Disease: Studies with Levetiracetam.

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 Aß was accompanied by improvement of synaptic function and plasticity. As the negative effects of Aß 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.

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells.

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.

Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.

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.

Determination of cocaine, cocaine metabolites and cannabinoids in single hairs by MALDI Fourier transform mass spectrometry--preliminary results.

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.

Adsorption of plasma proteins on uncoated PLGA nanoparticles.

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.

Perfusion reversed-phase high-performance liquid chromatography for protein separation from detergent-containing solutions: an alternative to gel-based approaches.

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.

Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score.

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.