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Background: The utility of magnetic resonance imaging (MRI) in the primary care setting is uncertain, with a perception that there is less likelihood for surgery after MRI ordered by general practitioners (GPs) when compared with orthopaedic surgeons and sports medicine physicians. Additionally, the influence of patient age and sex on subsequent surgical intervention is currently unknown., Purpose/hypothesis: The purpose of this study was to compare surgical incidence after MRI referrals by orthopaedic surgeons, GPs, and sports medicine physicians, including a subset analysis for GP patients based on type of approval given by the radiologist. The authors also wanted to explore the association of age and sex on subsequent surgical intervention. They hypothesized that surgical incidence after MRI ordered by orthopaedic surgeons and sports medicine physicians would be higher than after MRI ordered by GPs., Study Design: Cohort study; Level of evidence, 3., Methods: Knee MRI referrals by the 3 physician cohorts during May to December 2017 were assessed. For GP patients, the types of approval or recommendation from a radiologist were categorized. Subsequent surgical intervention status was then compared among referral groups up to 2 years after MRI. Associations of age and sex with surgical occurrence were also assessed. Chi-square test, analysis of variance, and univariate/multivariable logistic regression were used for statistical analysis., Results: Overall, 407 referrals were evaluated (GP, n = 173; orthopaedic, n = 176; sports medicine, n = 58). Surgical incidence was not significantly higher for orthopaedic and sports medicine than GP referrals at 3 months (10%, 3%, and 6%, respectively; P = .23), 6 months (20%, 17%, and 15%; P = .49), and 2 years (30%, 35%, and 24%; P = .25). Surgical incidence for GP patients was higher after discussion with a radiologist or when evaluating specific pathology on prior imaging versus less defined reasons (30.4% vs 15.7%, respectively; P = .03). Surgical incidence was lower for older patients (11% vs 31% for >60 years vs all other age groups combined; P = .002), and women were less likely to undergo surgery than men (22% vs 35%, respectively; P = .008)., Conclusion: Surgical incidence after MRI was likely appropriately lower for older patients. Lower incidence for female patients is of uncertain cause and warrants further study., Competing Interests: The authors have declared that there are no conflicts of interest in the authorship and publication of this contribution. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2021.)

Background: Postoperative rehabilitation after arthroscopic rotator cuff repair (ARCR) remains controversial and suffers from limited high-quality evidence. Therefore, appropriate use criteria must partially depend on expert opinion., Hypothesis/purpose: The purpose of the study was to determine and report on the standard and modified rehabilitation protocols after ARCR used by member orthopaedic surgeons of the American Orthopaedic Society for Sports Medicine (AOSSM) and the Arthroscopy Association of North America (AANA). We hypothesized that there will exist a high degree of variability among rehabilitation protocols. We also predict that surgeons will be prescribing accelerated rehabilitation., Study Design: Cross-sectional study; Level of evidence, 4., Methods: A 29-question survey in English language was sent to all 3106 associate and active members of the AOSSM and the AANA. The questionnaire consisted of 4 categories: standard postoperative protocol, modification to postoperative rehabilitation, operative technique, and surgeon demographic data. Via email, the survey was sent on September 4, 2013., Results: The average response rate per question was 22.7%, representing an average of 704 total responses per question. The most common immobilization device was an abduction pillow sling with the arm in neutral or slight internal rotation (70%). Surgeons tended toward later unrestricted passive shoulder range of motion at 6 to 7 weeks (35%). Strengthening exercises were most commonly prescribed between 6 weeks and 3 months (56%). Unrestricted return to activities was most commonly allowed at 5 to 6 months. The majority of the respondents agreed that they would change their protocol based on differences expressed in this survey., Conclusion: There is tremendous variability in postoperative rehabilitation protocols after ARCR. Five of 10 questions regarding standard rehabilitation reached a consensus statement. Contrary to our hypothesis, there was a trend toward later mobilization., Competing Interests: The authors declared that they have no conflicts of interest in the authorship and publication of this contribution.

The HUPO Human Proteome Project (HPP) has two overall goals: (1) stepwise completion of the protein parts list-the draft human proteome including confidently identifying and characterizing at least one protein product from each protein-coding gene, with increasing emphasis on sequence variants, post-translational modifications (PTMs), and splice isoforms of those proteins; and (2) making proteomics an integrated counterpart to genomics throughout the biomedical and life sciences community. PeptideAtlas and GPMDB reanalyze all major human mass spectrometry data sets available through ProteomeXchange with standardized protocols and stringent quality filters; neXtProt curates and integrates mass spectrometry and other findings to present the most up to date authorative compendium of the human proteome. The HPP Guidelines for Mass Spectrometry Data Interpretation version 2.1 were applied to manuscripts submitted for this 2016 C-HPP-led special issue [ www.thehpp.org/guidelines ]. The Human Proteome presented as neXtProt version 2016-02 has 16,518 confident protein identifications (Protein Existence [PE] Level 1), up from 13,664 at 2012-12, 15,646 at 2013-09, and 16,491 at 2014-10. There are 485 proteins that would have been PE1 under the Guidelines v1.0 from 2012 but now have insufficient evidence due to the agreed-upon more stringent Guidelines v2.0 to reduce false positives. neXtProt and PeptideAtlas now both require two non-nested, uniquely mapping (proteotypic) peptides of at least 9 aa in length. There are 2,949 missing proteins (PE2+3+4) as the baseline for submissions for this fourth annual C-HPP special issue of Journal of Proteome Research. PeptideAtlas has 14,629 canonical (plus 1187 uncertain and 1755 redundant) entries. GPMDB has 16,190 EC4 entries, and the Human Protein Atlas has 10,475 entries with supportive evidence. neXtProt, PeptideAtlas, and GPMDB are rich resources of information about post-translational modifications (PTMs), single amino acid variants (SAAVSs), and splice isoforms. Meanwhile, the Biology- and Disease-driven (B/D)-HPP has created comprehensive SRM resources, generated popular protein lists to guide targeted proteomics assays for specific diseases, and launched an Early Career Researchers initiative., Competing Interests: The authors declare no competing financial interest.

Large scale proteomics have made it possible to broadly screen samples for the presence of many types of post-translational modifications, such as phosphorylation, acetylation, and ubiquitination. This type of data has allowed the localization of these modifications to either a specific site on a proteolytically generated peptide or to within a small domain on the peptide. The resulting modification acceptor sites can then be mapped onto the appropriate protein sequences and the information archived. This paper describes the usage of a very large archive of experimental observations of human post-translational modifications to create a map of the most reproducible modification observations onto the complete set of human protein sequences. This set of modification acceptor sites was then directly translated into the genomic coordinates for the codons for the residues at those sites. We constructed the database g2pDB using this protein-to-codon site mapping information. The information in g2pDB has been made available through a RESTful-style API, allowing researchers to determine which specific protein modifications would be perturbed by a set of observed nucleotide variants determined by high throughput DNA or RNA sequencing.

The growing complexity of proteomics samples and the desire for deeper analysis drive the development of both better MS instrument and advanced multidimensional separation schemes. We applied 1D, 2D, and 3D LC-MS/MS separation protocols (all of reversed-phase C18 functionality) to a tryptic digest of whole Jurkat cell lysate to estimate the depth of proteome coverage and to collect high-quality peptide retention information. We varied pH of the eluent and hydrophobicity of ion-pairing modifier to achieve good separation orthogonality (utilization of MS instrument time). All separation modes employed identical LC settings with formic-acid-based eluents in the last dimension. The 2D protocol used a high pH-low pH scheme with 21 concatenated fractions. In the 3D protocol, six concatenated fractions from the first dimension (C18, heptafluorobutyric acid) were analyzed using the identical 2D LC-MS procedure. This approach permitted a detailed evaluation of the analysis output consuming 21× and 126× the analysis time and sample load compared to 1D. Acquisition over 189 h of instrument time in 3D mode resulted in the identification of ∼14 000 proteins and ∼250 000 unique peptides. We estimated the dynamic range via peak intensity at the MS(2) level as approximately 10(4.2), 10(5.6), and 10(6.2) for the 1D, 2D, and 3D protocols, respectively. The uniform distribution of the number of acquired MS/MS, protein, and peptide identifications across all 126 fractions and through the chromatographic time scale in the last LC-MS stage indicates good separation orthogonality. The protocol is scalable and is amenable to the use of peptide retention prediction in all dimensions. All these features make it a very good candidate for large-scale bottom-up proteomic runs, which target both protein identification as well as the collection of peptide retention data sets for targeted quantitative applications.

Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein-protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation.

The honey bee is a key pollinator in agricultural operations as well as a model organism for studying the genetics and evolution of social behavior. The Apis mellifera genome has been sequenced and annotated twice over, enabling proteomics and functional genomics methods for probing relevant aspects of their biology. One troubling trend that emerged from proteomic analyses is that honey bee peptide samples consistently result in lower peptide identification rates compared with other organisms. This suggests that the genome annotation can be improved, or atypical biological processes are interfering with the mass spectrometry workflow. First, we tested whether high levels of polymorphisms could explain some of the missed identifications by searching spectra against the reference proteome (OGSv3.2) versus a customized proteome of a single honey bee, but our results indicate that this contribution was minor. Likewise, error-tolerant peptide searches lead us to eliminate unexpected post-translational modifications as a major factor in missed identifications. We then used a proteogenomic approach with ~1500 raw files to search for missing genes and new exons, to revive discarded annotations and to identify over 2000 new coding regions. These results will contribute to a more comprehensive genome annotation and facilitate continued research on this important insect.

V-erb-b2 erythroblastic leukemia viral oncogene homologue 2, known as ERBB2, is an important oncogene in the development of certain cancers. It can form a heterodimer with other epidermal growth factor receptor family members and activate kinase-mediated downstream signaling pathways. ERBB2 gene is located on chromosome 17 and is amplified in a subset of cancers, such as breast, gastric, and colon cancer. Of particular interest to the Chromosome-Centric Human Proteome Project (C-HPP) initiative is the amplification mechanism that typically results in overexpression of a set of genes adjacent to ERBB2, which provides evidence of a linkage between gene location and expression. In this report we studied patient samples from ERBB2-positive together with adjacent control nontumor tissues. In addition, non-ERBB2-expressing patient samples were selected as comparison to study the effect of expression of this oncogene. We detected 196 proteins in ERBB2-positive patient tumor samples that had minimal overlap (29 proteins) with the non-ERBB2 tumor samples. Interaction and pathway analysis identified extracellular signal regulated kinase (ERK) cascade and actin polymerization and actinmyosin assembly contraction as pathways of importance in ERBB2+ and ERBB2- gastric cancer samples, respectively. The raw data files are deposited at ProteomeXchange (identifier: PXD002674) as well as GPMDB.

Remarkable progress continues on the annotation of the proteins identified in the Human Proteome and on finding credible proteomic evidence for the expression of "missing proteins". Missing proteins are those with no previous protein-level evidence or insufficient evidence to make a confident identification upon reanalysis in PeptideAtlas and curation in neXtProt. Enhanced with several major new data sets published in 2014, the human proteome presented as neXtProt, version 2014-09-19, has 16,491 unique confident proteins (PE level 1), up from 13,664 at 2012-12 and 15,646 at 2013-09. That leaves 2948 missing proteins from genes classified having protein existence level PE 2, 3, or 4, as well as 616 dubious proteins at PE 5. Here, we document the progress of the HPP and discuss the importance of assessing the quality of evidence, confirming automated findings and considering alternative protein matches for spectra and peptides. We provide guidelines for proteomics investigators to apply in reporting newly identified proteins.

Unlabelled: The Global Proteome Machine and Database (GPMDB) representational state transfer (REST) service was designed to provide simplified access to the proteomics information in GPMDB using a stable set of methods and parameters. Version 1 of this interface gives access to 25 methods for retrieving experimental information about protein post-translational modifications, amino acid variants, alternate splicing variants and protein cleavage patterns., Availability and Implementation: GPMDB data and database tables are freely available for commercial and non-commercial use. All software is also freely available, under the Artistic License. http://rest.thegpm.org/1 (GPMDB REST Service), http://wiki.thegpm.org/wiki/GPMDB_REST (Service description and help), and http://www.thegpm.org (GPM main project description and documentation). The code for the interface and an example REST client is available at ftp://ftp.thegpm.org/repos/gpmdb_rest, (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

One year ago the Human Proteome Project (HPP) leadership designated the baseline metrics for the Human Proteome Project to be based on neXtProt with a total of 13,664 proteins validated at protein evidence level 1 (PE1) by mass spectrometry, antibody-capture, Edman sequencing, or 3D structures. Corresponding chromosome-specific data were provided from PeptideAtlas, GPMdb, and Human Protein Atlas. This year, the neXtProt total is 15,646 and the other resources, which are inputs to neXtProt, have high-quality identifications and additional annotations for 14,012 in PeptideAtlas, 14,869 in GPMdb, and 10,976 in HPA. We propose to remove 638 genes from the denominator that are "uncertain" or "dubious" in Ensembl, UniProt/SwissProt, and neXtProt. That leaves 3844 "missing proteins", currently having no or inadequate documentation, to be found from a new denominator of 19,490 protein-coding genes. We present those tabulations and web links and discuss current strategies to find the missing proteins.

In this study we selected three breast cancer cell lines (SKBR3, SUM149 and SUM190) with different oncogene expression levels involved in ERBB2 and EGFR signaling pathways as a model system for the evaluation of selective integration of subsets of transcriptomic and proteomic data. We assessed the oncogene status with reads per kilobase per million mapped reads (RPKM) values for ERBB2 (14.4, 400, and 300 for SUM149, SUM190, and SKBR3, respectively) and for EGFR (60.1, not detected, and 1.4 for the same 3 cell lines). We then used RNA-Seq data to identify those oncogenes with significant transcript levels in these cell lines (total 31) and interrogated the corresponding proteomics data sets for proteins with significant interaction values with these oncogenes. The number of observed interactors for each oncogene showed a significant range, e.g., 4.2% (JAK1) to 27.3% (MYC). The percentage is measured as a fraction of the total protein interactions in a given data set vs total interactors for that oncogene in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.0) and I2D (Interologous Interaction Database, version 1.95). This approach allowed us to focus on 4 main oncogenes, ERBB2, EGFR, MYC, and GRB2, for pathway analysis. We used bioinformatics sites GeneGo, PathwayCommons and NCI receptor signaling networks to identify pathways that contained the four main oncogenes and had good coverage in the transcriptomic and proteomic data sets as well as a significant number of oncogene interactors. The four pathways identified were ERBB signaling, EGFR1 signaling, integrin outside-in signaling, and validated targets of C-MYC transcriptional activation. The greater dynamic range of the RNA-Seq values allowed the use of transcript ratios to correlate observed protein values with the relative levels of the ERBB2 and EGFR transcripts in each of the four pathways. This provided us with potential proteomic signatures for the SUM149 and 190 cell lines, growth factor receptor-bound protein 7 (GRB7), Crk-like protein (CRKL) and Catenin delta-1 (CTNND1) for ERBB signaling; caveolin 1 (CAV1), plectin (PLEC) for EGFR signaling; filamin A (FLNA) and actinin alpha1 (ACTN1) (associated with high levels of EGFR transcript) for integrin signalings; branched chain amino-acid transaminase 1 (BCAT1), carbamoyl-phosphate synthetase (CAD), nucleolin (NCL) (high levels of EGFR transcript); transferrin receptor (TFRC), metadherin (MTDH) (high levels of ERBB2 transcript) for MYC signaling; S100-A2 protein (S100A2), caveolin 1 (CAV1), Serpin B5 (SERPINB5), stratifin (SFN), PYD and CARD domain containing (PYCARD), and EPH receptor A2 (EPHA2) for PI3K signaling, p53 subpathway. Future studies of inflammatory breast cancer (IBC), from which the cell lines were derived, will be used to explore the significance of these observations.

We report progress assembling the parts list for chromosome 17 and illustrate the various processes that we have developed to integrate available data from diverse genomic and proteomic knowledge bases. As primary resources, we have used GPMDB, neXtProt, PeptideAtlas, Human Protein Atlas (HPA), and GeneCards. All sites share the common resource of Ensembl for the genome modeling information. We have defined the chromosome 17 parts list with the following information: 1169 protein-coding genes, the numbers of proteins confidently identified by various experimental approaches as documented in GPMDB, neXtProt, PeptideAtlas, and HPA, examples of typical data sets obtained by RNASeq and proteomic studies of epithelial derived tumor cell lines (disease proteome) and a normal proteome (peripheral mononuclear cells), reported evidence of post-translational modifications, and examples of alternative splice variants (ASVs). We have constructed a list of the 59 "missing" proteins as well as 201 proteins that have inconclusive mass spectrometric (MS) identifications. In this report we have defined a process to establish a baseline for the incorporation of new evidence on protein identification and characterization as well as related information from transcriptome analyses. This initial list of "missing" proteins that will guide the selection of appropriate samples for discovery studies as well as antibody reagents. Also we have illustrated the significant diversity of protein variants (including post-translational modifications, PTMs) using regions on chromosome 17 that contain important oncogenes. We emphasize the need for mandated deposition of proteomics data in public databases, the further development of improved PTM, ASV, and single nucleotide variant (SNV) databases, and the construction of Web sites that can integrate and regularly update such information. In addition, we describe the distribution of both clustered and scattered sets of protein families on the chromosome. Since chromosome 17 is rich in cancer-associated genes, we have focused the clustering of cancer-associated genes in such genomic regions and have used the ERBB2 amplicon as an example of the value of a proteogenomic approach in which one integrates transcriptomic with proteomic information and captures evidence of coexpression through coordinated regulation.

Experiments to probe for protein-protein interactions are the focus of functional proteomic studies, thus proteomic data repositories are increasingly likely to contain a large cross-section of such information. Here, we use the Global Proteome Machine database (GPMDB), which is the largest curated and publicly available proteomic data repository derived from tandem mass spectrometry, to develop an in silico protein interaction analysis tool. Using a human histone protein for method development, we positively identified an interaction partner from each histone protein family that forms the histone octameric complex. Moreover, this method, applied to the α subunits of the human proteasome, identified all of the subunits in the 20S core particle. Furthermore, we applied this approach to human integrin αIIb and integrin β3, a major receptor involved in the activation of platelets. We identified 28 proteins, including a protein network for integrin and platelet activation. In addition, proteins interacting with integrin β1 obtained using this method were validated by comparing them to those identified in a formaldehyde-supported coimmunoprecipitation experiment, protein-protein interaction databases and the literature. Our results demonstrate that in silico protein interaction analysis is a novel tool for identifying known/candidate protein-protein interactions and proteins with shared functions in a protein network.

Purpose: To determine the incidence of glenohumeral chondrolysis associated with the use of a continuous-infusion device in shoulder arthroscopy., Methods: A consecutive series of patients undergoing arthroscopic glenohumeral surgery with a postoperative continuous-infusion pump inserted into either the glenohumeral joint or subacromial space were evaluated for chondrolysis. Two pump types were used: group 1 received 100 mL of 0.5% bupivacaine without epinephrine infused at 2.08 mL/h, and group 2 received 270 mL of 0.5% bupivacaine without epinephrine infused at 4.16 mL/h., Results: We followed up 65 patients at a mean of 40 months. Of these, 29 had glenohumeral catheters (13 in group 1 and 16 in group 2) and 36 had subacromial catheters (19 in group 1 and 17 in group 2). The overall postoperative Constant, American Shoulder and Elbow Surgeons, Rowe, Single Assessment Numeric Evaluation, and Simple Shoulder Test scores were 84, 87, 77, 86, and 10, respectively, in those with glenohumeral catheters and 93, 94, 95, 89, and 11, respectively, in those with subacromial catheters. Three glenohumeral catheter patients were diagnosed with chondrolysis, all in group 2., Conclusions: Chondrolysis developed in 3 of 16 patients (19%) with glenohumeral joint infusion of 0.5% bupivacaine without epinephrine at 4.16 mL/h for 65 hours. No patient using a 2.08-mL/h reservoir for 48 hours into the glenohumeral joint and no patient with a subacromial infusion device had chondrolysis. Clinical symptoms and radiographic evidence of chondrolysis developed before 12 months after surgery., Level of Evidence: Level III, retrospective comparative study.

Acetylation is a well-studied posttranslational modification that has been associated with a broad spectrum of biological processes, notably gene regulation. Many studies have contributed to our knowledge of the enzymology underlying acetylation, including efforts to understand the molecular mechanism of substrate recognition by several acetyltransferases, but traditional experiments to determine intrinsic features of substrate site specificity have proven challenging. Here, we combine experimental methods with clustering analysis of protein sequences to predict protein acetylation based on the sequence characteristics of acetylated lysines within histones with our unique prediction tool PredMod. We define a local amino acid sequence composition that represents potential acetylation sites by implementing a clustering analysis of histone and nonhistone sequences. We show that this sequence composition has predictive power on 2 independent experimental datasets of acetylation marks. Finally, we detect acetylation for selected putative substrates using mass spectrometry, and report several nonhistone acetylated substrates in budding yeast. Our approach, combined with more traditional experimental methods, may be useful for identifying acetylated substrates proteome-wide.

Multiple reaction monitoring (MRM), commonly employed for the mass spectrometric detection of small molecules, is rapidly gaining ground in proteomics. Its high sensitivity and specificity makes this targeted approach particularly useful when sample throughput or proteome coverage limits global studies. Existing tools to design MRM assays rely exclusively on theoretical predictions, or combine them with previous observations on the same type of sample. The additional mass spectrometric experimentation this requires can pose significant demands on time and material. To overcome these challenges, a new MRM worksheet was introduced into The Global Proteome Machine database (GPMDB) that provided all of the information needed to design MRM transitions based solely on archived observations made by other researchers in previous experiments. This required replacing the precursor ion intensity by the number of peptide observations, which proved to be an adequate substitute if peptides did not occur in multiple forms. While the absence of collision energy information proved largely inconsequential, successful prediction of unique transitions depended on the type of fragment ion involved. The design of MRM assays for iTRAQ-labeled tryptic peptides obtained from human platelet proteins demonstrated the usefulness of the MRM worksheet also for quantitative applications. This workflow, which relies exclusively on experimental observations stored in data repositories, therefore represents an attractive alternative for the prediction of MRM transitions prior to experimental validation and optimization.

Purpose: To compare the biomechanical performance of several different sutures by evaluating knot security and load to failure strength using different arthroscopic knots., Methods: Eight different No. 2 sutures (Ethibond [Ethicon, Somerville, NJ], FiberWire [Arthrex, Naples, FL], Orthocord [DePuy-Mitek, Norwood, MA], Hi-Fi [ConMed Linvatec, Largo, FL], Ultrabraid [Smith & Nephew, Andover, MA], ForceFiber [Stryker Endoscopy, San Jose, CA], MagnumWire [ArthroCare, Sunnyvale, CA], and MaxBraid PE [Arthrotek, Warsaw, IN]) were tied arthroscopically into standardized loops using 6 different knots (Weston, Tennessee slider, Duncan, SMC, Revo, and San Diego knot) 10 times each. The suture loops were pretensioned to 10N, cycled between 10N and 45N for 1,000 cycles, and loaded to failure. The failure load for each suture, each knot, and slippage trend during cyclic loading was recorded., Results: The Revo and SMC knots (group A) were stronger than the Tennessee and San Diego knots (group B), which were stronger than the Weston knot, which was stronger than the Duncan loop (P < .05). This pattern also coincided with the loads at which these knots slipped. Evaluating the sutures showed that Ethibond had lower failure loads than all other sutures and FiberWire showed statistically higher loads (P < .05). Duncan loops (97.5%) and Weston knots (86.3%) slipped more than other knots (P < .001), while the SMC and Revo knots slipped least. Ethibond sutures were least likely to slip., Conclusions: The Duncan loop and Weston knot were more likely to slip than all other knots, and caution should be exercised when tying them with high-strength sutures. The Revo, Tennessee slider, San Diego, and SMC knots were least likely to slip (P < .001)., Clinical Relevance: While stronger than braided polyester sutures, newer sutures containing ultra-high molecular weight polyethylene have a greater tendency to slip. Backing up knots with 4 reversed half hitches with switched posts does not guarantee knot security.

Purpose: The purpose of this study was to evaluate the clinical success of the FasT-Fix meniscal repair device (Smith & Nephew Endoscopy, Andover, MA) associated with an accelerated rehabilitation program., Methods: A prospectively collected consecutive series of meniscal repairs performed with the FasT-Fix device was studied. The Lysholm, Tegner, Cincinnati, and International Knee Documentation Committee activity scores, along with the clinical examination findings and adverse events, were recorded for all patients. Associated procedures were recorded. An accelerated postoperative rehabilitation program was followed, independent of concurrent anterior cruciate ligament surgery., Results: Forty-one meniscal repairs were performed, with an average follow-up of 30.7 months (range, 12 to 58 months). Twenty-nine of 41 repairs were performed in conjunction with anterior cruciate ligment reconstruction. The other repairs were in stable knees. There were 26 medial and 15 lateral meniscus repairs. Both menisci were repaired in 5 knees. Repeat arthroscopies were performed for 12 repairs and 7 (17%) were found to have failed. The preoperative and postoperative Lysholm, Tegner, Cincinnati, and International Knee Documentation Committee activity scores were 47.3 and 87.4, 3.4 and 7.2, 38.7 and 82.8, and 2.3 and 3.4, respectively., Conclusions: The FasT-Fix meniscal repair associated with an accelerated rehabilitation program resulted in clinically effective meniscal repair in 83% at the time of follow-up. Clinical outcome measures all improved., Level of Evidence: Level IV, therapeutic case series.

Purpose: Our purpose was to evaluate recently introduced sutures and suture anchors for single pull load to failure strength and failure mode., Methods: Suture anchors were tested in fresh porcine metaphyseal cortex and cancellous troughs using an established protocol. An Instron machine applied tensile loads parallel to the axis of insertion at a rate of 12.5 mm per second until failure and mean anchor failure strengths were calculated. The mode of failure was recorded (anchor pullout, suture eyelet cut out, or suture failure). Anchors tested included the Kinsa, Kinsa RC, BioRaptor 2.3 PK, TwinFix PK FT 5.5 and 6.5, BioCleat, Healix Peek, VersaLok, BioKnotless, BioKnotless BR, Corkscrew FT III, SwiveLock C, and PEEK SutureTak., Results: The mean cortical failure loads were as follows: Kinsa (219 N), Kinsa RC (222 N), BioRaptor 2.3 PK (172 N), TwinFix PK FT 5.5 (491 N) and 6.5 (503 N), BioCleat (218 N), Healix Peek (407 N), VersaLok (376 N), BioKnotless (249 N), BioKnotless BR (265 N), Corkscrew FT III (386 N), SwiveLock C (712 N), and PEEK SutureTak (168 N). Pullout was the predominant failure mode for the VersaLok, BioKnotless, BioKnotless BR, and BioRaptor 2.3PK anchors. Eyelet failure was the predominant failure mode for the Kinsa, Kinsa RC, BioCleat, Healix Peek, Corkscrew FT III, SwiveLock C, and PEEK SutureTak., Conclusions: The newer anchors showed markedly increased load to failure strengths. Two or more high-strength sutures are commonly used as well as new anchor materials (PEEK and Biocryl Rapide), new eyelet designs, and the increased use of a "knotless" concept., Clinical Relevance: An anchor which fails principally by pull out at a low load to failure is at risk for creating an intra-articular loose body.

Analyzer-based imaging (ABI) utilizes synchrotron radiation sources to create collimated monochromatic x-rays. In addition to x-ray absorption, this technique uses refraction and scatter rejection to create images. ABI provides dramatically improved contrast over standard imaging techniques. Twenty-one adult male Wistar rats were divided into four experimental groups to undergo the following interventions: (1) non-injured control, (2) decortication alone, (3) decortication with iliac crest bone grafting and (4) decortication with iliac crest bone grafting and interspinous wiring. Surgical procedures were performed at the L5-6 level. Animals were killed at 2, 4 and 6 weeks after the intervention and the spine muscle blocks were excised. Specimens were assessed for the presence of fusion by (1) manual testing, (2) conventional absorption radiography and (3) ABI. ABI showed no evidence of bone fusion in groups 1 and 2 and showed solid or possibly solid fusion in subjects from groups 3 and 4 at 6 weeks. Metal artifacts were not present in any of the ABI images. Conventional absorption radiographs did not provide diagnostic quality imaging of either the graft material or fusion masses in any of the specimens in any of the groups. Synchrotron-based ABI represents a novel imaging technique which can be used to assess spinal fusion in a small animal model. ABI produces superior image quality when compared to conventional radiographs.

Analyses of proteins from a number of proteomic studies of cell membranes have demonstrated that a significant component of the identified proteins is not predicted to contain transmembrane regions. The presence of such proteins may arise as a result of contamination of the membrane preparations or through real associations. Our aim was to identify integral proteins as well as those that are intimately associated with the microsomal membranes of K562 cells. Isolated membranes were treated under conditions reported to remove noncovalently associated 'peripheral' proteins and the residual proteins were SDS-PAGE-separated and analyzed by LC-MS/MS. Tandem lectin affinity was also examined as a complementary approach for the enrichment of membrane glycoproteins. Approximately 41% of the isolated proteins were assigned as membrane proteins based on the presence of transmembrane regions or covalent post-translational modifications that could account for membrane association. Collectively, these results indicate that there is a significant component of non integral proteins that appear to be as closely associated with membranes as integral elements.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been successfully applied to elucidating biological questions trough the analysis of proteins, peptides, and nucleic acids. Here, we review the different approaches for analyzing the data that is generated by MALDI-MS. The first step in the analysis is the processing of the raw data to find peaks that correspond to the analytes. The peaks are characterized by their areas (or heights) and their centroids. The peak area can be used as a measure of the quantity of the analyte, and the centroid can be used to determine the mass of the analyte. The masses are then compared to models of the analyte, and these models are ranked according to how well they fit the data and their significance is calculated. This allows the determination of the identity (sequence and modifications) of the analytes. We show how this general data analysis workflow is applied to protein and nucleic acid chemistry as well as proteomics., ((c) 2007 Wiley Periodicals, Inc.)

Background: Diffraction-enhanced imaging (DEI) is a synchrotron-based x-ray imaging technique that has dramatically improved contrast over standard x-ray imaging techniques. It is possible to acquire images that analyze the x-ray refraction and the apparent absorption (elimination of small-angle scattering) of the object., Methods: Three formalin-fixed porcine eyes were studied at the National Synchrotron Light Source using DEI. Conventional absorption-type radiography was conducted for comparison., Results: Conventional absorption radiography did not yield significant detail of the eye anatomy. DEI showed excellent characterization of many ocular structures. The cornea, iris, lens, retina, optic nerve, as well as choroidal vasculature and the ampullae of the vortex veins could all be visualized., Interpretation: DEI represents a novel, high-resolution imaging technique that has excellent characterization of ocular anatomy. Further application of this imaging modality will be undertaken to study cataract and choroidal tumors and to examine ocular surface structures, such as the extraocular muscle insertions, more closely.

This paper described a simple heuristic method for determining the merit of a set of peptide sequence assignments made using tandem mass spectra. The method involved comparing a prediction based on the known stochastic behavior of a sequence assignment algorithm with the assignments generated from a particular data set. A particular formulation of this comparison was defined through the construction of a plot of the data, the rho-diagram, as well as a parameter derived from this plot, the rho-score. This plot and parameter were shown to be able to readily characterize the relative quality of a set of peptide sequence assignments and to allow the straightforward determination of probability threshold values for the interpretation of proteomics data. This plot is independent of the algorithm or scoring scheme used to estimate the statistical significance of a set of experimental results; rather, it can be used as an objective test of the correctness of those estimates. The rho-score can also be used as a parameter to evaluate the relative merit of protein identifications, such as those made across proteome species taxonomic categories.

Fabry disease is inherited as an X-linked disorder secondary to deficiency of alpha-galactosidase A, resulting in abnormal metabolism of substances containing alpha-d-galactosyl moieties. As a consequence, a multisystem disorder develops, culminating in strokes, progressive renal, and cardiac dysfunction. Signs and symptoms of Fabry disease become manifest in childhood, but diagnosis is often delayed. Thirteen children with Fabry disease (age range, 6.5-17 years) were studied as part of a 6-month open-label study of enzyme replacement therapy (ERT) with agalsidase alfa. Paired serum samples were drawn at the start of the study and after 6 months of ERT. Global protein changes in paired samples were compared by using differential stable isotope labeling of peptide lysine residues with O-methylisourea and subsequent nanoHPLC-tandem MS. Statistically significant decreases were observed for five proteins following ERT: alpha(2)-HS glycoprotein, vitamin D-binding protein, transferrin, Ig-alpha-2 C chain, and alpha-2-antiplasmin. The presence of low levels of alpha-2-antiplasmin and plasminogen was confirmed by alternate means in 34 consecutive patients, including four of five ERT-naïve subjects. Decreased alpha-2-antiplasmin was associated with a parallel increase in circulating VEGF. Soluble VEGF receptor-2 was significantly elevated in plasma of patients compared with pediatric controls and decreased with ERT. These results suggest previously unknown abnormalities of fibrinolysis and angiogenesis factors in Fabry disease. We demonstrated the feasibility of identifying treatment-specific alterations in a small number of subjects that point to previously unsuspected disease-related biological abnormalities.

Motivation: Tandem mass spectrometry (MS/MS) identifies protein sequences using database search engines, at the core of which is a score that measures the similarity between peptide MS/MS spectra and a protein sequence database. The TANDEM application was developed as a freely available database search engine for the proteomics research community. To extend TANDEM as a platform for further research on developing improved database scoring methods, we modified the software to allow users to redefine the scoring function and replace the native TANDEM scoring function while leaving the remaining core application intact. Redefinition is performed at run time so multiple scoring functions are available to be selected and applied from a single search engine binary. We introduce the implementation of the pluggable scoring algorithm and also provide implementations of two TANDEM compatible scoring functions, one previously described scoring function compatible with PeptideProphet and one very simple scoring function that quantitative researchers may use to begin their development. This extension builds on the open-source TANDEM project and will facilitate research into and dissemination of novel algorithms for matching MS/MS spectra to peptide sequences. The pluggable scoring schema is also compatible with related search applications P3 and Hunter, which are part of the X! suite of database matching algorithms. The pluggable scores and the X! suite of applications are all written in C++., Availability: Source code for the scoring functions is available from http://proteomics.fhcrc.org

Introduction: Diffraction-enhanced imaging (DEI) uses monochromatic synchrotron X-rays to image tissue. This technique has been shown to produce superior bony and soft tissue characterization when compared with conventional absorption radiography. Application of this imaging modality is under investigation, and this study represents the first DEI analysis of the vertebral column., Methods: Four male Wistar rats were studied. Spine muscle blocks were imaged in 3 of the rats after thoracic laminectomy (n = 1), after lumbar laminectomy (n = 1), and in a control condition (n = 1). The fourth rat was imaged as a whole animal control. Conventional radiography and synchrotron-supported DEI at 40 keV were performed on all specimens. We compared images side by side, using a nonvalidated subjective assessment technique., Results: DEI produced superior visualization of the vertebral anatomy, compared with conventional absorption radiography for all specimens. Greater bony and soft tissue detail was noted, with improved image contrast. In addition to imaging the anatomical structures, DEI showed the polyglactin suture material used for fascial closure in the 2 animals that underwent surgery. Artifact from air bubbles was present on DEI images but not on plain radiographs., Conclusions: This represents the first use of DEI, a novel imaging modality, to image the vertebral column. It provides excellent anatomic detail with superior contrast and visualization of both bone and soft tissue when compared with conventional radiography. Future applications of this investigational technique may include analysis of spinal fusion as well as degenerative and neoplastic conditions of the spine.

A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-A pore size columns. Correlations of up to approximately 0.98 R2 value were obtained for a test library of approximately 2000 peptides and approximately 0.95-0.97 for a variety of real samples. The algorithm was applied in conjunction with an exclusion protocol based on mass (15 ppm tolerance) and retention time (2-min tolerance for 0.66% acetonitrile/min gradient), MART criteria to significantly reduce the instrument time required for complete MS/MS analysis of a digest separated by RP HPLC. This was confirmed by reanalyzing the set of HPLC-MALDI MS/MS data with no loss in protein identifications, despite the number of virtually executed MS/MS analyses being decreased by 57%.

A system for creating a library of tandem mass spectra annotated with corresponding peptide sequences was described. This system was based on the annotated spectra currently available in the Global Proteome Machine Database (GPMDB). The library spectra were created by averaging together spectra that were annotated with the same peptide sequence, sequence modifications, and parent ion charge. The library was constructed so that experimental peptide tandem mass spectra could be compared with those in the library, resulting in a peptide sequence identification based on scoring the similarity of the experimental spectrum with the contents of the library. A software implementation that performs this type of library search was constructed and successfully used to obtain sequence identifications. The annotated tandem mass spectrum libraries for the Homo sapiens, Mus musculus, and Saccharomyces cerevisiae proteomes and search software were made available for download and use by other groups.

The environment and equipment used in shop class are potential sources of serious injury. There has been little published to date on injuries sustained in shop class, with no reports examining injuries to the hand. The authors report a case series collected from a health records database at a pediatric and hand surgery referral center. Fifteen patients who sustained injuries to their wrist or hand in shop class were identified. Sixty percent of the injuries were caused by table saws. Eighty percent required treatment from a hand surgeon. Sixty-seven percent of patients sustained a serious injury in the form of amputation or tendon or neurovascular injury. Most of the patients had functional deficits at final follow-up. Shop class is a setting where serious hand trauma can occur. School administrators and educators should direct efforts at preventing these injuries. Parents and students must recognize the risks associated with shop class. Physicians should be prepared for severe injuries and the frequent need for hand surgical consultation.

This chapter describes the use of an open-source, freely available informatics system for the identification of proteins using tandem mass spectra of peptides derived from an enzymatic digest of a mixture of mature proteins. The chapter describes the use of features of the Global Proteome Machine (GPM) interface that assist in making comprehensive assignments between spectra and sequences, including the detection of point mutations, posttranslational modifications, and experimental artifacts. The use of this interface to validate results using the GPM Database is also described. This data repository allows analysts to compare their own results to those obtained by other scientists to determine the degree to which their data are consistent with previous measurements.

Background: Spontaneous spinal epidural hematoma is a rare phenomenon that has no distinct etiology. Spontaneous spinal epidural hematoma (SSEH) during pregnancy is extremely rare. We present what we believe to be the fifth reported case of spontaneous spinal epidural hematoma associated with pregnancy in the English literature., Methods: A 31-year-old female presented with acute onset of paraplegia at 32 weeks of pregnancy. The patient had a T2 sensory level and complete paralysis of all lower extremity motor groups. Magnetic resonance imaging of the thoracic spine showed an acute epidural hematoma posterior to the thoracic spinal cord between the second and fourth thoracic vertebrae., Results: The patient was taken to the operating room were her child was delivered by caesarean section. She then underwent a posterior laminectomy and evacuation of a spinal epidural hematoma. Follow-up selective spinal angiography was negative for any vascular malformation. The patient gradually recovered lower extremity function and was independently ambulating at six month follow-up. Voluntary bowel and bladder function returned within four months but twice daily intermittent catheterization remained necessary for excessive post-void residual urine., Conclusions: Spontaneous spinal epidural hematoma in pregnancy is a rare phenomenon. It is postulated that elevated venous pressure associated with pregnancy may be a contributing factor. In the reported cases of SSEH in pregnancy most patients presented with acute symptoms, thoracic location and profound neurological deficits but, with prompt surgical treatment, generally had good long term recovery.

This paper describes an algorithm to apply proteotypic peptide sequence libraries to protein identifications performed using tandem mass spectrometry (MS/MS). Proteotypic peptides are those peptides in a protein sequence that are most likely to be confidently observed by current MS-based proteomics methods. Libraries of proteotypic peptide sequences were compiled from the Global Proteome Machine Database for Homo sapiens and Saccharomyces cerevisiae model species proteomes. These libraries were used to scan through collections of tandem mass spectra to discover which proteins were represented by the data sets, followed by detailed analysis of the spectra with the full protein sequences corresponding to the discovered proteotypic peptides. This algorithm (Proteotypic Peptide Profiling, or P3) resulted in sequence-to-spectrum matches comparable to those obtained by conventional protein identification algorithms using only full protein sequences, with a 20-fold reduction in the time required to perform the identification calculations. The proteotypic peptide libraries, the open source code for the implementation of the search algorithm and a website for using the software have been made freely available. Approximately 4% of the residues in the H. sapiens proteome were required in the proteotypic peptide library to successfully identify proteins., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

This paper describes an open-source system for analyzing, storing, and validating proteomics information derived from tandem mass spectrometry. It is based on a combination of data analysis servers, a user interface, and a relational database. The database was designed to store the minimum amount of information necessary to search and retrieve data obtained from the publicly available data analysis servers. Collectively, this system was referred to as the Global Proteome Machine (GPM). The components of the system have been made available as open source development projects. A publicly available system has been established, comprised of a group of data analysis servers and one main database server.

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.

The aim was to determine the proportion of membrane glycoproteins captured using concanavalin A or wheat germ agglutinin lectin affinity chromatography. Digests of the isolated proteins were separated by reversed-phase liquid chromatography and analyzed by matrix-assisted laser desorption tandem mass spectrometry. The two lectins identified different groups of proteins with a broad range of molecular mass and p/ values, including a number of proteins that overlapped the two groups. Approximately 30% of the proteins were positively identified as containing domains that were predicted using standard bioinformatics methods to be characteristic of integral membrane proteins. This approach represents an effective method of surveying the membrane protein pool of mammalian cells for subsequent proteomic analysis.

Summary: Tandem mass spectra obtained from fragmenting peptide ions contain some peptide sequence specific information, but often there is not enough information to sequence the original peptide completely. Several proprietary software applications have been developed to attempt to match the spectra with a list of protein sequences that may contain the sequence of the peptide. The application TANDEM was written to provide the proteomics research community with a set of components that can be used to test new methods and algorithms for performing this type of sequence-to-data matching., Availability: The source code and binaries for this software are available at http://www.proteome.ca/opensource.html, for Windows, Linux and Macintosh OSX. The source code is made available under the Artistic License, from the authors.

The paper describes the implementation of a software system based on the Fenyö disulfide bond assignment algorithm. The system allows an investigator to enter data derived from mass spectrum peak assignments, a target protein sequence and other experimental conditions. The output of the system is the set of disulfide bonding pattern models that are consistent with the experimental evidence. The software and code are available through a public web site, which also has a functioning, publicly accessible version of the disulfide bond modeler. This implementation was tested as part of a project to check homology-based assignments disulfide bonding patterns of human integrins.

This paper investigates the use of survival functions and expectation values to evaluate the results of protein identification experiments. These functions are standard statistical measures that can be used to reduce various protein identification scoring schemes to a common, easily interpretably representation. The relative merits of scoring systems were explored using this approach, as well as the effects of altering primary identification parameters. We would advocate the widespread use of these simple statistical measures to simplify and standardize the reporting of the confidence of protein identification results, allowing the users of different identification algorithms to compare their results in a straightforward and statistically significant manner. A method is described for measuring these distributions using information that is being discarded by most protein identification search engines, resulting in accurate survival functions that are specific to any combination of scoring algorithms, sequence databases, and mass spectra.

An algorithm for reducing the time necessary to match a large set of peptide tandem mass spectra with a list of protein sequences is described. This algorithm breaks the process into multiple steps. A rapid survey step identifies all protein sequences that are reasonable candidates for a match with a set of tandem mass spectra. These candidates are then used as models, which are refined by detailed analysis of the set of tandem mass spectra for evidence of incomplete enzymatic hydrolysis, non-specific hydrolysis and chemical modifications of amino acid residues resulting from either post-translational modifications or sample handling. Compared with current one-step methods for matching proteins to mass spectra, this multiple-step method can decrease the time required for the calculation by several orders of magnitude., (Copyright 2003 John Wiley & Sons, Ltd.)

Proteomics has become dominated by large amounts of experimental data and interpreted results. This experimental data cannot be effectively used without understanding the fundamental structure of its information content and representing that information in such a way that knowledge can be extracted from it. This review explores the structure of this information with regard to three fundamental issues: the extraction of relevant information from raw data, the scale of the projects involved and the statistical significance of protein identification results.

RADARS, a rapid, automated, data archiving and retrieval software system for high-throughput proteomic mass spectral data processing and storage, is described. The majority of mass spectrometer data files are compatible with RADARS, for consistent processing. The system automatically takes unprocessed data files, identifies proteins via in silico database searching, then stores the processed data and search results in a relational database suitable for customized reporting. The system is robust, used in 24/7 operation, accessible to multiple users of an intranet through a web browser, may be monitored by Virtual Private Network, and is secure. RADARS is scalable for use on one or many computers, and is suited to multiple processor systems. It can incorporate any local database in FASTA format, and can search protein and DNA databases online. A key feature is a suite of visualisation tools (many available gratis), allowing facile manipulation of spectra, by hand annotation, reanalysis, and access to all procedures. We also described the use of Sonar MS/MS, a novel, rapid search engine requiring 40 MB RAM per process for searches against a genomic or EST database translated in all six reading frames. RADARS reduces the cost of analysis by its efficient algorithms: Sonar MS/MS can identifiy proteins without accurate knowledge of the parent ion mass and without protein tags. Statistical scoring methods provide close-to-expert accuracy and brings robust data analysis to the non-expert user.

Hypertrophy is an adaptive response of the heart to myocardial injury or hemodynamic overload that may progress and contribute to cardiac decompensation and eventually to heart failure. The signaling pathways controlling this response in the cardiac myocyte are poorly understood. A data mining effort of a human failed heart cDNA library was undertaken in an effort to identify novel signaling molecules involved in cardiac hypertrophy. This effort identified a novel kinase (MLK7) homologous to the mixed lineage kinase family of proteins. The mixed lineage kinases are mitogen-activated protein kinase kinase kinases (MAPKKKs) which activate stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 kinase pathways. They contain a catalytic domain with homology to both serine/threonine and tyrosine-specific kinases and a dual leucine zipper. MLK7 is identical to leucine zipper and sterile-alpha motif protein kinase (ZAK) through the leucine zipper domain but has a completely divergent COOH-terminus and shares approximately 40% homology with the other MLKs overall. Expression of MLK7 mRNA is most abundant in skeletal muscle and heart, with expression restricted to the cardiac myocyte. The recombinant histidine tagged MLK7 expressed and purified from insect cells exhibited serine/threonine kinase activity in vitro with myelin basic protein as substrate. When expressed in cardiac myocytes, MLK7 activated SAPK/JNK1, and ERK and p38 to a lesser extent. Additionally, MLK7 altered fetal gene expression and increased protein synthesis in cardiac myocytes. These data suggest that MLK7 is a new member of the mixed lineage kinase family that modulates cardiac SAPK/JNK pathway and may play a role in cardiac hypertrophy and progression to heart failure., (Copyright 2001 Academic Press.)

Gerstmann-Sträussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Sträussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.