Max Planck Institute of Biochemistry

The PRoteomics IDEntifications (PRIDE) database (https://www.ebi.ac.uk/pride/) is the world's largest data repository of mass spectrometry-based proteomics data, and is one of the founding members of the global ProteomeXchange (PX) consortium. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2016. In the last 3 years, public data sharing through PRIDE (as part of PX) has definitely become the norm in the field. In parallel, data re-use of public proteomics data has increased enormously, with multiple applications. We first describe the new architecture of PRIDE Archive, the archival component of PRIDE. PRIDE Archive and the related data submission framework have been further developed to support the increase in submitted data volumes and additional data types. A new scalable and fault tolerant storage backend, Application Programming Interface and web interface have been implemented, as a part of an ongoing process. Additionally, we emphasize the improved support for quantitative proteomics data through the mzTab format. At last, we outline key statistics on the current data contents and volume of downloads, and how PRIDE data are starting to be disseminated to added-value resources including Ensembl, UniProt and Expression Atlas.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

A key step in mass spectrometry (MS)-based proteomics is the identification of peptides in sequence databases by their fragmentation spectra. Here we describe Andromeda, a novel peptide search engine using a probabilistic scoring model. On proteome data, Andromeda performs as well as Mascot, a widely used commercial search engine, as judged by sensitivity and specificity analysis based on target decoy searches. Furthermore, it can handle data with arbitrarily high fragment mass accuracy, is able to assign and score complex patterns of post-translational modifications, such as highly phosphorylated peptides, and accommodates extremely large databases. The algorithms of Andromeda are provided. Andromeda can function independently or as an integrated search engine of the widely used MaxQuant computational proteomics platform and both are freely available at www.maxquant.org. The combination enables analysis of large data sets in a simple analysis workflow on a desktop computer. For searching individual spectra Andromeda is also accessible via a web server. We demonstrate the flexibility of the system by implementing the capability to identify cofragmented peptides, significantly improving the total number of identified peptides.

Protein quantification without isotopic labels has been a long-standing interest in the proteomics field. However, accurate and robust proteome-wide quantification with label-free approaches remains a challenge. We developed a new intensity determination and normalization procedure called MaxLFQ that is fully compatible with any peptide or protein separation prior to LC-MS analysis. Protein abundance profiles are assembled using the maximum possible information from MS signals, given that the presence of quantifiable peptides varies from sample to sample. For a benchmark dataset with two proteomes mixed at known ratios, we accurately detected the mixing ratio over the entire protein expression range, with greater precision for abundant proteins. The significance of individual label-free quantifications was obtained via a t test approach. For a second benchmark dataset, we accurately quantify fold changes over several orders of magnitude, a task that is challenging with label-based methods. MaxLFQ is a generic label-free quantification technology that is readily applicable to many biological questions; it is compatible with standard statistical analysis workflows, and it has been validated in many and diverse biological projects. Our algorithms can handle very large experiments of 500+ samples in a manageable computing time. It is implemented in the freely available MaxQuant computational proteomics platform and works completely seamlessly at the click of a button.

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Efficient folding of many newly synthesized proteins depends on assistance from molecular chaperones, which serve to prevent protein misfolding and aggregation in the crowded environment of the cell. Nascent chain--binding chaperones, including trigger factor, Hsp70, and prefoldin, stabilize elongating chains on ribosomes in a nonaggregated state. Folding in the cytosol is achieved either on controlled chain release from these factors or after transfer of newly synthesized proteins to downstream chaperones, such as the chaperonins. These are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. Understanding how the thousands of different proteins synthesized in a cell use this chaperone machinery has profound implications for biotechnology and medicine.

Viroids are uncoated infectious RNA molecules pathogenic to certain higher plants. Four different highly purified viroids were studied. By ultracentrifugation, thermal denaturation, electron microscopy, and end group analysis the following features were established: (i) the molecular weight of cucumber pale fruit viroid from tomato is 110,000, of citrus exocortis viroid from Gynura 119,000, of citrus exocortis viroid from tomato 119,000 and of potato spindle tuber viroid from tomato 127,000. (ii) Viroids are single-stranded molecules. (iii) Virods exhibit high thermal stability, cooperativity, and self-complementarity resulting in a rod-like native structure. (iv) Viroids are covalently closed circular RNA molecules.

We have isolated a large noncollagenous glycoprotein, laminin, from a mouse tumor that produces basement membrane. The protein consists of at least two polypeptide chains (Mr = 220,000 and Mr = 440,000) joined to each other by disulfide bonds. Laminin and type IV collagen are major constituents of the tumor. Laminin is distinctly different from fibronectin, another component of basement membranes, in amino acid composition and immunological reactivity. Pepsin digestion of laminin releases a large, cystine-rich fragment which retains most of the antigenicity of the original protein. Immunological studies using purified antibody against laminin show that it is produced by a variety of cultured cells. In addition, these antibodies react with the basement membranes of normal tissues, suggesting that this protein or an immunologically related protein is a constituent of the basement membranes of these tissues.

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.

Directed movement is a characteristic of many living organisms and occurs as a result of the transformation of chemical energy into mechanical energy. Myosin is one of three families of molecular motors that are responsible for cellular motility. The three-dimensional structure of the head portion of myosin, or subfragment-1, which contains both the actin and nucleotide binding sites, is described. This structure of a molecular motor was determined by single crystal x-ray diffraction. The data provide a structural framework for understanding the molecular basis of motility.

There is increasing evidence that neurotrophins (NTs) are involved in processes of neuronal plasticity besides their well-established actions in regulating the survival, differentiation, and maintenance of functions of specific populations of neurons. Nerve growth factor, brain-derived neurotrophic factor, NT-4/5, and corresponding antibodies dramatically modify the development of the visual cortex. Although the neuronal elements involved have not yet been identified, complementary studies of other systems have demonstrated that NT synthesis is rapidly regulated by neuronal activity and that NTs are released in an activity-dependent manner from neuronal dendrites. These data, together with the observation that NTs enhance transmitter release from neurons that express the corresponding signal-transducing Trk receptors, suggest a role for NTs as selective retrograde messengers that regulate synaptic efficacy.

In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. The 26S proteasome is a 2.5-MDa molecular machine built from approximately 31 different subunits, which catalyzes protein degradation. It contains a barrel-shaped proteolytic core complex (the 20S proteasome), capped at one or both ends by 19S regulatory complexes, which recognize ubiquitinated proteins. The regulatory complexes are also implicated in unfolding and translocation of ubiquitinated targets into the interior of the 20S complex, where they are degraded to oligopeptides. Structure, assembly and enzymatic mechanism of the 20S complex have been elucidated, but the functional organization of the 19S complex is less well understood. Most subunits of the 19S complex have been identified, however, specific functions have been assigned to only a few. A low-resolution structure of the 26S proteasome has been obtained by electron microscopy, but the precise arrangement of subunits in the 19S complex is unclear.

Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.

Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MSn spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the “lock mass” by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification. Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MSn spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the “lock mass” by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification. The data produced by a mass spectrometer are the mass and intensity of compounds and their fragments. The accuracy of mass measurement directly determines the usefulness of mass spectrometric experiments, and much effort in instrumentation development is directed at improving this key parameter. Mass accuracy and mass resolution are connected, and instruments introduced during the last decades radically improved in these two attributes. Traditionally accurate mass measurements, sufficient to determine the elemental composition of small molecules, were the province of magnetic sector instruments, but today TOF instruments equipped with energy correcting reflectrons can reach low ppm values. Triple quadrupole instruments or quadrupole ion traps, which are popular in proteomics research, however, have low resolution and mass uncertainties of typically half to several Da. At the other extreme, FT-ICR mass spectrometers reduce the mass measurement to a frequency measurement and are therefore potentially capable of exceedingly high mass accuracy. In practice, however, FT-ICR instruments have suffered from the requirement to precisely control the number of ions accumulated in the Penning trap. Over or under filling leads to mass shifts to high and low values, respectively. For example, Smith and co-workers (1Belov M.E. Zhang R. Strittmatter E.F. Prior D.C. Tang K. Smith R.D. Automated gain control and internal calibration with external ion accumulation capillary liquid chromatography-electrospray ionization Fourier transform ion cyclotron resonance.Anal. Chem. 2003; 75: 4195-4205Google Scholar) reported that in their measurements the mass determined over an LC peak varied by more than 10 ppm. The recent introduction of a linear ion trap-FT-ICR combination (2Syka J.E.P. Marto J.A. Bai D.L. Horning S. Senko M.W. Schwartz J.C. Ueberheide B. Garcia B. Busby S. Muratore T. Shabanowitz J. Hunt D.F. Novel Linear Quadrupole Ion Trap/FT Mass Spectrometer: Performance Characterization and Use in the Comparative Analysis of Histone H3 Post-translational Modifications.J. Proteome Res. 2004; 3: 621-626Google Scholar) largely solved this problem through a prescan in the ion trap to estimate ion current (called automatic gain control), which allows filling of the ICR cell with a predetermined number of ions. Using automatic gain control and narrow mass ranges (SIM 1The abbreviations used are: SIM, selected ion monitoring; MS/MS, tandem MS; LTQ, Thermo Electron linear quadrupole ion trap; RF, radio frequency; SILAC, stable isotope labeling by amino acids in cell culture; PCM, polycyclodimethylsiloxane. scans) we observed an average absolute mass error between 0.6 and 0.7 ppm in recent large scale proteomic analyses (3Olsen J.V. Ong S.E. Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues.Mol. Cell Proteomics. 2004; 3: 608-614Google Scholar, 4Andersen J.S. Lam Y.W. Leung A.K. Ong S.E. Lyon C.E. Lamond A.I. Mann M. Nucleolar proteome dynamics.Nature. 2005; 433: 77-83Google Scholar, 5Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell Proteomics. 2005; 4: 310-327Google Scholar). In a typical proteomics experiment, protein mixtures are digested to peptide mixtures that are separated by reversed phase HPLC and analyzed on-line by MS and MS/MS (6Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Google Scholar). The mass accuracy achieved in the instrument directly translates into the mass tolerances that can be specified in subsequent database searches of tandem mass spectra. Unambiguous protein identification in large data sets is by no means trivial (7Steen H. Mann M. The abc’s (and xyz’s) of peptide sequencing.Nat. Rev. Mol. Cell. Biol. 2004; 5: 699-711Google Scholar), and any increase in achieved mass accuracy greatly aids the specificity of database searches in two ways (8Jensen O.N. Podtelejnikov A. Mann M. Delayed Extraction Improves Specificity in Database Searches by MALDI Peptide Maps.Rapid Commun. Mass Spectrom. 1996; 10: 1371-1378Google Scholar, 9Clauser K.R. Baker P. Burlingame A.L. Role of accurate mass measurement (+/− 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.Anal. Chem. 1999; 71: 2871-2882Google Scholar): High precursor mass accuracy in the MS spectra directly translates into fewer “candidate sequences” that need to be considered as possible matches. High mass accuracy in the MS/MS spectra leads to fewer measured fragment masses that match the calculated fragments of a candidate sequence by chance and therefore decreases the scores of false positives in database search algorithms. In 1923, Kingdon (10Kingdon K. A method for the neutralization of electron space charge by positive ionization at very low gas pressures.Phys. Rev. 1923; 21: 408-418Google Scholar) devised a method to capture ions by causing them to orbit around a central electrode. Since then, the physics community has used “Kingdon traps” in a variety of experiments, but it was always used as a capturing device, not as a mass spectrometer. A few years ago a mass which the orbitrap A. a of mass Chem. Scholar, M. A. the orbitrap mass to an ion Chem. 2003; 75: Scholar, H. A. M. R. The a mass Mass Spectrom. 2005; Scholar). the of this a mass spectrometer was very introduced of the linear ion trap coupled to a radio frequency for storage of ions and of the orbitrap mass (LTQ and S. of Mass in Mass Spectrom. Scholar). In to the Penning used in the orbitrap of two around which injected ions magnetic are and the of the is a few The is an current of the of ion the and the mass spectrum is as the Fourier transform of this a the has high and it be capable of high mass accuracy. mass accuracy on the of an which is more to achieve than of a magnetic of the mass accuracy of an orbitrap for proteomic have reported Here we that very high mass accuracy is possible on the a background ion produced by in ambient and a number of this ion into the the trap the to the The ions the mass spectrum are then added to this “lock and all are injected into the orbitrap average the mass over the of peptide these average absolute mass in the sub-ppm for at The masses of to the are determined to a few ppm. of was in a 2 and and digested as Mann M. proteomics of high specificity for signaling A. 2003; Scholar). reduce was added to a of 10 in the protein and for at in the The were with for at The and protein mixtures were digested and the peptide mixtures were on as J. Mann M. and for and in Chem. 2003; 75: Scholar) and in for analysis. The which has a and which is therefore an for was from of yeast were in yeast liquid or for 10 of the and yeast determined by were then by for at at two with by and for protein Cell were by in a The yeast was to the was transferred to a and the protein in the was determined by were separated by and to the The was with the were and digested with were into and with For protein were with 10 in for at of was by the with in for at in the were two with with and in a The were with in and for at for protein were transferred to and the remaining were by two with in by with The were and and the used for mass spectrometric analysis. digested peptide mixtures were separated by on-line and analyzed by tandem mass The were on an to an mass spectrometer equipped with a ion and of the in a from with The peptide mixtures were injected the with a of and with a of from in were for and for the yeast The mass spectrometer was in the to between and MS spectra were in the orbitrap with resolution at accumulation to a of in the linear ion The ions to on signal were for in the linear ion trap at a of The fragment ions were in the orbitrap with resolution at For accurate mass measurements the mass was in MS and MS/MS and the ions in the from ambient air A. R. in the ambient air as of background signals in mass Mass Spectrom. 2003; Scholar) were used for internal recalibration in For of the mass into the the mass was at of the of the mass The mass time orbitrap and subsequent spectra was in The time for the and into the of the mass was to be a few at no time in with and mass was in MS/MS the ion at with was used for ions selected for MS/MS were for mass spectrometric no and gas ion gas for Ion was for and time of was applied for were by database an of the protein sequence database or of the for yeast database was with observed and specified an MS tolerance of 10 ppm and an MS/MS tolerance at possible in and specificity for to 2 of was as a and of and were as variable of the high mass the in the yeast database search was a of even into the high fragment mass and with a greater than this were for for peptide mass due to imperfect peaks caused by weak signals we have a that all MS mass measurements of a peptide ion over the LC peak, weighted by signal intensity. For any data all peak and charge of the mass to the peptide is in the measured peptide masses are over the weighted by its signal intensity in The was in and with Thermo Electron data the a in which all peptide ion masses have with the are in and peak for the yeast in the are in the of the orbitrap mass spectrometer have in the A. a of mass Chem. Scholar, M. A. the orbitrap mass to an ion Chem. 2003; 75: Scholar, H. A. M. R. The a mass Mass Spectrom. 2005; Scholar), we the for of the mass accuracy addressed can be in it of The is a capable of MS and MSn spectra at very high but low resolution and mass accuracy. Ions accumulated in the can be transferred into a an quadrupole that and the ions. The to the of the in the of the In the ions are by a low of and to in the of the trap. are injected into the orbitrap high with few Ions are then in the orbitrap by the of the with of the ion Ions to around the and into that the in The of is determined by the of ion from the orbitrap of is by the of injected ion The frequency of this is to the of the mass to charge of the ions and is by a to of the the For a a is produced and the signals of an ion can be into a mass spectrum by Fourier ions are to in the and therefore the the and In the can be with a not with the of the mass of 10 and the of the which has not determined but which is than the of the C-trap. is used as a gas it is used in the of the instrument and it is at capturing and the ions. In the the linear ion trap and fragments it be possible to fragment ions in the it is more and to them in the linear ion trap and in the C-trap. employing the linear ion trap as a the well low mass for fragments to the as The mass of the to in the will be the as in the in is to ion J.C. Senko M.W. A quadrupole ion trap mass Mass Spectrom. Scholar, K. of a ion trap and a ion trap mass spectrometer in Cell Proteomics. 2005; 4: the orbitrap as a high mass in the we injected of a of a a and measured MS and MS/MS spectra with the orbitrap as the were ions for MS and ions for MS/MS in the Analysis of fragments was in the the of the can be with a between acquisition and high to resolution at and MS/MS to a resolution of time for by MS/MS for to of the ions was on from orbitrap accumulation for MS and for and the ion a of a typical mass High and signal to are of the a typical MS/MS spectrum with resolution and a of The that charge of fragment ions is trivial the high resolution and peak in the LTQ, the can to fragments in the or in the orbitrap can be in the the of all ions are as the ion is more in the spectrum, that this into the at the in The between and MS/MS spectra with that on an mass ranges were than and that it was to mass ranges to a MS/MS spectrum J. and M. of high low mass accuracy MS/MS for in Mass Spectrom. Scholar). is by the of ion caused by over the from the to the and from the to the of a spectrum of a peptide ion current of the MS/MS spectrum of a peptide in the and analyzed in the of the ion at Da. that the isotope it to the charge of MS/MS analyzed in the orbitrap in the by 10 The the MS/MS spectrum in the ion in the orbitrap was very as in the observed that of were sufficient to signal to MS/MS spectra. In this experiment, were of the of have even were not for as as the signal is at the of the the as a we of or to filling that are in the of mixtures the is typically by the time in acquisition of the than the ion as was the in this we observed that MS/MS spectra in the orbitrap are less than spectra in the is caused by the high resolution of the orbitrap and its current At ions of the are for and S. of Mass in Mass Spectrom. therefore background ions from which are not to in the spectra. The of MS/MS spectra by in the orbitrap will the acquisition therefore orbitrap MS/MS will be to MS/MS we to the mass accuracy on the mass accuracy was we observed with caused by ambient can be in that mass is much than mass which by a few parts per In mass a mass is to for and to an internal in the spectrum the of For example, was measured in the of a to a sub-ppm mass accuracy Mass for in MALDI Chem. 1999; 71: Scholar), and with a for and internal in mass C.E. calibration on with Fourier transform mass Chem. Scholar). used a ion of which has a composition of and an mass of and is in spectra A. R. in the ambient air as of background signals in mass Mass Spectrom. 2003; Scholar). in a of a few to an a number of background which can all be used as masses for internal calibration during analysis. in mass accuracy of the peptide improved from ppm to ppm by employing as In mass spectrum, we the mass to calibration and all measured by the ppm deviation for all masses as for the the measured mass of the background ion as a of The observed mass is stable less than ppm during time but in the time of spectrum of ambient a of 2 to an in the ambient of these ions can be used as masses during of of the measured mass of a peptide the mass of the background ion the of a mass is well it is not is in to the effort of a mass into the Here we PCM, which is always during the it is not always in or spectra peptide signals to the background ion signals to we of a of the its C-trap. A number of ions used a of is accumulated in the and transferred to the C-trap. In this an signal of is always of the accumulation time used for the mass number of ions not the of the C-trap. of ions and the and no more than a few to the the mass can be added to any For example, ions of can be accumulated in the and a precursor ion can be accumulated and in the LTQ, which is by of all MS/MS ions into the C-trap. of an mass or of masses has into the data and is the mass signal is from the spectra. we the of mass for the of a peptide S.E. B. H. A. Mann M. by in Cell SILAC, as a and to Cell Proteomics. Scholar) yeast was digested with and analyzed by on the The acquisition was as in mass for all spectra. identification of yeast by database mass between calculated and measured peptide masses were in that the of mass is with all or two ppm. the of the few at mass we the mass deviation as a of peptide intensity. that the are mainly caused by low and S. of Mass in Mass Spectrom. Scholar) have reported that signal to is a of achieved mass accuracy in orbitrap mass which is in with on other instruments C.E. Scholar). The data that the mass can reduce the mass error to a few ppm on the of the mass measurement that is the for peptide for improve we of the that several mass measurements are of the precursor as it from the capillary and that these measurements typically are of intensity than the used for precursor the intensity of a yeast peptide as it from the can be in the the precursor was for and its mass determined it was less than of its maximal intensity. mass measurements of the precursor were and it is from that mass accuracy is much at the of the LC peak than it is at its we a to over the LC which the mass measurements weighted by signal intensity. the of these mass accuracy was improved and was a ppm absolute deviation from the calculated values. that all the have a maximal mass error of less than 2 ppm average mass accuracy of 0.48 ppm and a deviation of 0.38 the mass can be applied for MS/MS spectra. however, that the ion of in the during the time it to and fragment the precursor we used this ion for mass scale a and of two peptide spectra in time with the mass spectra were with a of and a resolution of at the mass accuracy to be in the MS/MS with the MS of the and resolution as well as the that a tandem mass spectrum was for all fragments 2 ppm of their calculated provided that the intensity was than with less than were ppm of the calculated in all that the elemental composition of low mass ions can be determined a mass accuracy of a that can be in of tandem mass for example, to peptide sequence M. of in by Peptide Chem. Scholar), sequence B. M. Mann M. Peptide by MALDI tandem mass Proteome Res. Scholar), data sequence of peptide MS/MS data and the of MS/MS Cell Proteomics. 2005; 4: Scholar, protein identification in Fourier mass Cell Proteomics. 2005; 4: Scholar), or in composition B. peptide composition and a employing accurate mass by transform ion cyclotron mass Mass Spectrom. 2004; Scholar). orbitrap MS/MS spectra with the database the achieved mass accuracy is much than can be specified as a search parameter. the not the for mass than or scores to fragment with high mass accuracy J.S. protein identification by sequence mass 1999; Scholar). the the in to the peptide was very large with these high mass accuracy tandem mass in for spectra no peptide sequence was at on the can be in and 2 search with and LC mass and with peptide with and mass Here we a for very high mass accuracy with an mass spectrometer. a background ion of composition into the we for in the over The of this that the mass scale of the that is the of the frequency and of the is at to than per that the remaining mass error mainly on the signal intensity of the peptide For any peaks that are not to the we a mass accuracy to ppm. for weak peaks the mass accuracy is a few ppm. increase the mass we mass measurements over the LC peak weighted by signal intensity. In this several mass measurements to the and the mass is not on a signal to the as is the the precursor mass is the as the which is the for peak for of achieved mass accuracy on signal or signal to is not to the in current proteomic practice, a is The precursor and fragment mass tolerances are to even the mass mass have by recalibration J.S. A. Mann M. Analysis of the proteome by mass Scholar) or by mass as a be to very mass tolerances for well peaks and mass tolerances for weak a be in a all have with a mass In this with signal but large deviation from the calculated masses be from or at a precursor mass measurement be by its as A. A.I. R. to estimate the accuracy of peptide by MS/MS and database Chem. 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B. for Peptide Characterization by Mass Chem. 1996; Scholar) have that a mass accuracy of ppm peptide to a few and greatly peptide identification. Smith and co-workers added a time and that mass and time be sufficient for peptide identification E.F. Tang K. Smith R.D. Proteome analyses accurate mass and time peptide with capillary LC mass Mass Spectrom. 2003; Scholar). has to mass achieved to have for example, M.E. Smith R.D. A proteomic of the Proteome an accurate mass and time 2005; 5: Scholar). with the accuracy reported we not that the mass is sufficient to in typical proteomic the introduced by and peptide for as of the number of candidate peptide will be very and even low accuracy tandem mass spectra can then the candidate peptide is this mass accuracy will be in the of which a problem of the caused of S.E. Mann M. and in by 2004; Scholar). 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The three-dimensional structure of the proteasome from the archaebacterium Thermoplasma acidophilum has been elucidated by x-ray crystallographic analysis by means of isomorphous replacement and cyclic averaging. The atomic model was built and refined to a crystallographic R factor of 22.1 percent. The 673-kilodalton protease complex consists of 14 copies of two different subunits, alpha and beta, forming a barrel-shaped structure of four stacked rings. The two inner rings consist of seven beta subunits each, and the two outer rings consist of seven alpha subunits each. A narrow channel controls access to the three inner compartments. The alpha 7 beta 7 beta 7 alpha 7 subunit assembly has 72-point group symmetry. The structures of the alpha and beta subunits are similar, consisting of a core of two antiparallel beta sheets that is flanked by alpha helices on both sides. The binding of a peptide aldehyde inhibitor marks the active site in the central cavity at the amino termini of the beta subunits and suggests a novel proteolytic mechanism.

Eukaryotic cells replicate by a complex series of evolutionarily conserved events that are tightly regulated at defined stages of the cell division cycle. Progression through this cycle involves a large number of dedicated protein complexes and signaling pathways, and deregulation of this process is implicated in tumorigenesis. We applied high-resolution mass spectrometry-based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics. Co-regulated proteins and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global activation during S phase of the DNA damage response network, which was mediated by phosphorylation by ATM or ATR or DNA-dependent protein kinases. We determined site-specific stoichiometry of more than 5000 sites and found that most of the up-regulated sites phosphorylated by cyclin-dependent kinase 1 (CDK1) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells.

BACKGROUND Proteins are synthesized on ribosomes as linear chains of amino acids and must fold into unique three-dimensional structures to fulfill their biological functions. Protein folding is intrinsically error-prone, and how it is accomplished efficiently represents a problem of great biological and medical importance. During folding, the nascent polypeptide must navigate a complex energy landscape. As a result, misfolded molecules may accumulate that expose hydrophobic amino acid residues and thus are in danger of forming potentially toxic aggregates. To ensure efficient folding and prevent aggregation, cells in all domains of life express various classes of proteins called molecular chaperones. These proteins receive the nascent polypeptide chain emerging from the ribosome and guide it along a productive folding pathway. Because proteins are structurally dynamic, constant surveillance of the proteome by an integrated network of chaperones and protein degradation machineries, the proteostasis network (PN), is required to maintain protein homeostasis in a range of external and endogenous stress conditions. ADVANCES Over the past decade, we have gained substantial new insight into the overall behavior of the PN and the molecular mechanics of its components. Advances in structural biology and biophysical approaches have allowed chaperone mechanisms to be interrogated at an unprecedented level of detail. Recent work has provided fascinating insight into the process of protein folding on the ribosome and revealed how highly allosteric chaperones such as the heat shock protein 70 (Hsp70), Hsp90, and chaperonin systems modulate the folding energy landscapes of their protein clients. Studies of chaperone systems from bacteria and eukaryotes have revealed common principles underlying the organization of chaperone networks in different domains of life. Recently, we have begun to appreciate the relative complexity of eukaryotic chaperones and are starting to understand how eukaryotes deal with the challenge of folding a large proteome enriched in multidomain proteins. At the cellular level, the response of the PN to conformational stress, aging, and diseases of aberrant protein folding has been an area of intense investigation. Importantly, the capacity of the PN declines during aging and this leads to dysfunction of specific cell types and tissues, rendering the organism susceptible to chronic diseases. Among these, neurodegenerative syndromes associated with protein aggregation are increasingly prevalent in the aging human population. Notably, the accumulation of toxic protein aggregates is both a consequence and a cause of PN decline, driving a vicious cycle that ultimately leads to proteostasis collapse. OUTLOOK A new view of protein folding is emerging, whereby the energy landscapes that proteins navigate during folding in vivo may differ substantially from those observed during refolding in vitro. From the ribosome through to the major chaperone systems, the nascent protein interacts with factors that modulate its folding pathway. Future work should focus on obtaining the high-resolution structural and kinetic information necessary to define the pathways of protein folding during translation, and in association with molecular chaperones. Organisms have evolved various mechanisms to deal with misfolded and aggregated proteins to maintain proteostasis. It is becoming increasingly clear that besides removing these proteins by degradation, cells also strategically sequester them into transient or stable aggregates, often in defined cellular locations. Much remains to be understood about how this cellular decision-making occurs at a molecular level and how dysregulation of these mechanisms leads to proteotoxicity. From a medical perspective, the intimate relationship between proteostasis and disease, aging, and neurodegeneration makes components of the PN logical drug targets, with the goal of promoting healthy aging. Pharmacological manipulation of the PN will require a detailed understanding of how the network responds to perturbation and how its different components cooperate. Molecular chaperones are key players in the cellular proteostasis network and serve to maintain a balanced proteome. They promote the folding of newly synthesized proteins, function in conformational maintenance, and prevent potentially toxic off-pathway aggregation. Chaperones also cooperate with other components of the proteostasis network, such as the proteasome system and autophagy, in the removal of terminally misfolded and aggregated proteins through proteolytic degradation.

Electrons in correlated insulators are prevented from conducting by Coulomb repulsion between them. When an insulator-to-metal transition is induced in a correlated insulator by doping or heating, the resulting conducting state can be radically different from that characterized by free electrons in conventional metals. We report on the electronic properties of a prototypical correlated insulator vanadium dioxide in which the metallic state can be induced by increasing temperature. Scanning near-field infrared microscopy allows us to directly image nanoscale metallic puddles that appear at the onset of the insulator-to-metal transition. In combination with far-field infrared spectroscopy, the data reveal the Mott transition with divergent quasi-particle mass in the metallic puddles. The experimental approach used sets the stage for investigations of charge dynamics on the nanoscale in other inhomogeneous correlated electron systems.

The biological functions of proteins are governed by their three-dimensional fold. Protein folding, maintenance of proteome integrity, and protein homeostasis (proteostasis) critically depend on a complex network of molecular chaperones. Disruption of proteostasis is implicated in aging and the pathogenesis of numerous degenerative diseases. In the cytosol, different classes of molecular chaperones cooperate in evolutionarily conserved folding pathways. Nascent polypeptides interact cotranslationally with a first set of chaperones, including trigger factor and the Hsp70 system, which prevent premature (mis)folding. Folding occurs upon controlled release of newly synthesized proteins from these factors or after transfer to downstream chaperones such as the chaperonins. Chaperonins are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. This review focuses on recent advances in understanding the mechanisms of chaperone action in promoting and regulating protein folding and on the pathological consequences of protein misfolding and aggregation.