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Improving Peptide and Protein Identification Rates Using a Novel Semi-Supervised Approach in Scaffold (2013)
Protein Cluster Identification and Quantitation with Scaffold (2013)
Using MS MS Total Ion Current (TIC) Quantification in Scaffold 3 Software - Comparison to Spectral Counting (2011)

Probabilistically Assigning Sites of Protein Modification with Scaffold PTM (2011)

Testing for Differences Between Complex Samples in Proteomics Datasets (2009)

Quantitation Across Multiple iTRAQ Samples Using Scaffold Q+ (2009)

Improving Sensitivity by Combining MS MS Results (2007)

The Effect of Data Analysis Choices on the Number of Proteins Identified in MS MS Proteomics Experiments (2006)

Improving Computer Interpretation of Linear Ion Trap Data Using Scaffold (2006)

Identifying Peptides the Way Experts Do: A Scientist-Driven Scoring System for Improving MS MS Peptide Identification Confidence (2006)

Use of Multiple Peptide Identification Programs to Increase Peptide and Protein Identifications in Human Whole Saliva (2006)

Scaffold: A Program to Probabilistically Combine Results from Multiple MS MS Database Search Engines (2005)

Scaffold Citations

"Autoimmune response to transthyretin in juvenile idiopathic arthritis"
JCI insight (2016).
Cristina C. Clement, Halima Moncrieffe, Aditi Lele, Ginger Janow, Aniuska Becerra, Francesco Bauli, Fawzy A. Saad, Giorgio Perino, Cristina Montagna, Neil Cobelli, John Hardin, Lawrence J. Stern, Norman Ilowite, Steven A. Porcelli, and Laura Santambrogio

"Role of Carbonyl Modifications on Aging-Associated Protein Aggregation"
Nat. Scientific Reports (2016).
Maya Tanase, Aleksandra M. Urbanska, Valerio Zolla, Cristina C. Clement, Liling Huang, Kateryna Morozova, Carlo Follo, Michael Goldberg, Barbara Roda, Pierluigi Reschiglian & Laura Santambrogio

"Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly."
Nat. Cell Biol. (2015).
Horton ER, Byron A, Askari JA, Ng DH, Millon-Frémillon A, Robertson J, Koper EJ, Paul NR, Warwood S, Knight D, Humphries JD, Humphries MJ.

"Genetic background is a key determinant of glomerular extracellular matrix composition and organization"
J. Am. Soc. Nephrol. (2015)
J. Am. Soc. Nephrol. (2015), alternative link
Randles MJ, Woolf AS, Huang JL, Byron A, Humphries JD, Price KL, Kolatsi-Joannou M, Collinson S, Denny T, Knight D, Mironov A, Starborg T, Korstanje R, Humphries MJ, Long DA, Lennon R.

"Defining the phospho-adhesome through the phosphoproteomic analysis of integrin signalling"
Nat. Commun. (2015) 6: 6265
Nat. Commun. (2015) 6: 6265, alternative link
Robertson J, Jacquemet G, Byron A, Jones MC, Warwood S, Selley JN, Knight D, Humphries JD, Humphries MJ.

"A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting"
Nat. Commun. (2015) 6: 6135.
Nat. Commun. (2015) 6: 6135, alternative link
Byron A, Askari JA, Humphries JD, Jacquemet G, Koper EJ, Warwood S, Choi CK, Stroud MJ, Chen CS, Knight D, Humphries MJ.

"Microtubule-dependent modulation of adhesion complex composition"
PLoS One (2014) 9(12): e115213.
PLoS One (2014) 9(12): e115213, alternative link
Ng DH, Humphries JD, Byron A, Millon-Frémillon A, Humphries MJ.

"Global analysis reveals the complexity of the human glomerular extracellular matrix"
J. Am. Soc. Nephrol. (2014) 25(5): 939-51.
J. Am. Soc. Nephrol. (2014) 25(5): 939-51, alternative link
Lennon R, Byron A, Humphries JD, Randles MJ, Carisey A, Murphy S, Knight D, Brenchley PE, Zent R, Humphries MJ.

"Comparison of Fractionation Strategies Post-Enrichment by Titania used in the Study of the Phosphoproteome of a Green Alga"
Naldrett M.J., Marsh E.L., Alvarez S. and Evans B.S.
ABRF 2014 Waters Award Poster

“Circulating pEGFR is a Candidate Response Biomarker of Cetuximab Therapy in Colorectal Cancer”
Katsila T, Juliachs M, Gregori J, Macarulla T, Villarreal L,, Bardelli A, Torrance C, Elez, E, Tabernero J and Villanueva J*.
Clinical Cancer Research. 2014, 20(24):6346-56 .

“Enhancing the Biological Relevance of Secretome-based Proteomics by Linking Tumor Cell Proliferation and Protein Secretion”
Gregori J, Méndez O, Katsila T, Pujals M, Salvans C, Villarreal L, Arribas J, Tabernero J, Sánchez A, Villanueva J*.
J Proteome Research. 2014. 13 (8), 3706–3721.

"Linking the transcriptome and proteome to characterize the venom of the eastern diamondback rattlesnake (Crotalus adamanteus)"
Margres M.J., McGivern J.J., Wray K.P., Seavy M.,Calvin K. and Rokyta D.R.
Journal of Proteomics Volume 96, 16 January 2014, Pages 145–158

"Rac1 is deactivated at integrin activation sites through an IQGAP1-filamin-A-RacGAP1 pathway"
J. Cell Sci. (2013) 126(18): 4121-35.
J. Cell Sci. (2013) 126(18): 4121-35, alternative link
Jacquemet G, Morgan MR, Byron A, Humphries JD, Choi CK, Chen CS, Caswell PT, Humphries MJ.

“An effect size filter improves the reproducibility in spectral counting-based comparative proteomics”

"Proteomic anatomy of human skin"
“Unconventional secretion is a major contributor of cancer cell line secretomes”
"Bovine milk proteome: Quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis."

"Investigating the molecular mechanisms of the interactions between Lactobacillus reuteri strains and intestinal mucus."

"Batch effects correction improves the sensitivity of significance tests in spectral counting-based comparative discovery proteomics”

"Bovine milk exosome proteome."

"Shotgun proteomics-based clinical testing for diagnosis and classification of amyloidosis."
"Proteomics of human plasma: A critical comparison of analytical workflows in terms of effort, throughput and outcome."

"Comparative proteomic analysis of supportive and unsupportive extracellular matrix substrates for human embryonic stem cell maintenance."

"Proteomic analysis of extracellular matrix from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a as novel constituents of fibrotic liver."
"Proteomic analysis of α4β1 integrin adhesion complexes reveals α-subunit-dependent protein recruitment"

"Effects of Space Flight on the Expression of Liver Proteins in the Mouse"

"A Face in the Crowd: Recognizing Peptides Through Database Search"

"Proteomic Analysis of Integrin Adhesion Complexes"

"Protein identification using Sorcerer 2 and SEQUEST"

“Proteomic Analysis of Integrin-Associated Complexes Identifies RCC2 as a Dual Regulator Rac1 Arf6”

“Extensive fractionation and identification of proteins within nasal lavage fluids from allergic rhinitis and asthmatic chronic rhinosinusitis patients”

“Pathway-based biomarker search by high-throughput proteomics profiling of secretomes"

Temporal Quantitative Proteomics by iTRAQ 2D-LC-MS/MS and Corresponding mRNA Expression Analysis Identify Post-Transcriptional Modulation of Actin-Cytoskeleton Regulators During TGF-β-Induced Epithelial-Mesenchymal Transition”
  • J. Proteome Res., 2009, 8 (1), pp 35–47.
  • Venkateshwar G. Keshamouni, Pratik Jagtap, George Michailidis, John R. Strahler, Rork Kuick, Ajaya Kumar Reka, Panagiotis Papoulias, Rashmi Krishnapuram, Anjaiah Srirangam, Theodore J. Standiford, Philip C. Andrews and Gilbert S. Omenn

“Chicken egg yolk cytoplasmic proteome, mined via combinatorial peptide ligand libraries”

“Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes”

“Combining Results from Lectin Affinity Chromatography and Glycocapture Approaches Substantially Improves the Coverage of the Glycoproteome”

“Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage”

“Alterations in protein expression associated with the development of mealiness in peaches”

“Correlating the Transcriptome, Proteome, and Metabolome in the Environmental Adaptation of a Hyperthermophile”

“Identification of Fructose-1,6-bisphosphate aldolase cytosolic class I as an NMH7 MADS domain associated protein”

“Identification of Differentially Expressed Proteins in the Cervical Mucosa of HIV-1-Resistant Sex Workers”

“Proteomic Analysis of Rat Striatal Synaptosomes during Acrylamide Intoxication at a Low Dose Rate”

"Intracellular Trafficking of a Polymorphism in the Carboxyl Terminus of the Alpha Subunit of the Human Epithelial Sodium Channel is Modulated by Casein Kinase”

"Proteomic analysis of cervical-vaginal fluid: identification of novel biomarkers for detection of intra-amniotic infection"
  • J Proteome Res. 2007 Jan;6(1):89-96.
  • Gravett MG, Thomas A, Schneider KA, Reddy AP, Dasari S, Jacob T, Lu X, Rodland M, Pereira L, Sadowsky DW, Roberts CT Jr, Novy MJ, Nagalla SR.

"Global Assessment of Regulation of Phosphorylation of Insulin Receptor Substrate-1 by Insulin In Vivo in Human Muscle"

"Proteomic Analysis of human amniotic fluid"

"Quantitative phospho-proteomics of early elicitor signalling in Arabidopsis"

"Pfnek3 functions as an atypical MAPKK in Plasmodium falciparum "

"Proteomics Evaluation of Chemically Cleavable Activity-based Probes"

"Proteomic analysis of ovomucoid hypersensitivity in mice by two-dimensional difference gel electrophoresis (2D-DIGE)"

"Structure-Toxicity Analysis of Type-2 Alkenes: In Vitro Neurotoxicity"

"High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease"

"Tandem affinity purification of functional TAP-tagged proteins from human cells"

"Molecular Cloning and Expression of a cDNA Encoding a Coccidioides posadasii Cu,Zn Superoxide Dismutase Identified by Proteomic Analysis of the Coccidioidal T27K Vaccine"

"Proteomic analysis of conditioned media from three breast cancer cell lines: A mine for biomarkers and therapeutic targets"

"Endogenous phosphotyrosine signaling in zebrafish embryos"

"Characterization of the human skeletal muscle proteome by one-dimensional Gel electrophoresis and HPLC-ESI-MS/MS"

"Suction blister fluid as potential body fluid for biomarker proteins"
  • Proteomics, 7(20) 3638-3650.
  • Jeroen Kool, Léon Reubsaet, Feikje Wesseldijk, Raquel T. Maravilha, Martijn W. Pinkse, Clive S. D'Santos, Jacobus J. van Hilten, Freek J. Zijlstra, Albert J. R. Heck

"Proteomic profiling of human retinal and choroidal endothelial cells reveals molecular heterogeneity related to tissue of origin"

"Cannabinoid WIN 55,212-2 Regulates TRPV1 Phosphorylation in Sensory Neurons"

"Analysis of the cGMP/cAMP Interactome Using a Chemical Proteomics Approach in Mammalian Heart Tissue Validates Sphingosine Kinase Type 1-interacting Protein as Genuine and Highly Abundant AKAP"

"The Anti-HIV-1 Editing Enzyme APOBEC3G Binds HIV-1 RNA and Messenger RNAs That Shuttle between Polysomes and Stress Granules "

"Disease State Differentiation and Identification of Tuberculosis Biomarkers via Native Antigen Array Profiling"

"Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells"

"Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4, a component of the moving junction in Toxoplasma gondii."

"Identification of Hsc70 binding sites in mitochondrial aspartate aminotransferase."

"Lumbar Cerebrospinal Fluid Proteome in Multiple Sclerosis: Characterization by Ultrafiltration, Liquid Chromatography, and Mass Spectrometry"

"Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?"

"Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles."

Links & Other Resources

Search Engines and Other Programs

  • SEQUEST: SEQUEST is the original, and still one of the leading, programs that correlate uninterpreted MS/MS spectra of peptides with the amino acid sequences from protein databases. If you want to understand what SEQUEST is telling you, look at the interactive Sequest Browser summary page. SEQUEST is the search engine sold by Thermo Finnigan with its mass spectrometers. Sorcerer, a special hardware platform for running an optimized version of SEQUEST, is available from Sage N Research. DtaSelect is a program that organizes Sequest results. The Wildcat Toolbox from the Arizona Proteomics Consortium has various perl programs for further organizing Sequest, X! Tandem and FASTA files.
  • Mascot: Mascot is a widely used program that also uses mass spectrometry data to identify proteins from protein databases. Mascot has a extensive set of web help pages that includes a lot of information on protein identification by MS. You can use Mascot, in a limited way, on the web for free.
  • X!Tandem: X!Tandem is a very fast seach engine which searches for matches of MS/MS spectra to a protein database. X!Tandem is free to use on the web or to download. X!Tandem works in conjunction with and can take advantage of the information gleaned from previous searches. It has an option, PPP, which searches first only the peptides that have been found most frequently for each protein. This can speed searches up by a factor of 20.
  • Spectrum Mill: Spectrum Mill is the peptide search engine that Agilent sells with its mass spectrometers. Spectrum Mill implements a workflow for the MS experiment. First Spectrum Mill does an assessment of the quality of the spectra and only searches the worthwhile spectra. It also validates the spectra both automatically and visually.
  • ProteinLynx and IdentityE: ProteinLynx is the peptide search engine that Waters Micromass sells with its mass spectrometers. ProteinLynx manages a workflow which passes spectra not identified in the first search to a second or third search. For example the first search might be unmodified proteins, the second proteins with modifications and the third de novo sequencing followed by BLAST.
  • de novo - Peaks: Peaks takes MS/MS spectra files and does de novo sequencing to directly figure out what the amino acid sequence of the peptide is. Lutefisk and PepNovo are well regarded de novo sequencing programs that are available for free. De novo sequencing is also a part of Spectrum Mill and ProteinLynx and is also available from Thermo as denovoX.
  • Fragment Ion Calculator: If you have a peptide's sequence of amino acids, and want to see what the y and b ions are, use the Fragment Ion Calculator or Ion Predictor. This is useful if you want to understand the peaks that are labeled by SEQUEST or one of the other search engines.
  • Protein Digestor: The standard MS/MS experiment starts by digesting the proteins into peptides with an enzyme such as trypsin. To see what peptides will result from digesting a protein, use the Protein Digestor or the equivalent here. You can download a similar program, Paws.
  • ProSightPTM: In top down proteomics, an alternative way of gaining insight into a proteome, whole proteins are run through a mass spectrometer. ProSightPTM is the only software available for top down protein identification and characterization.
  • **GenoLogics LIMS**: A proteomics experiment involves much more than just mass spectrometry. There are also numerous steps of sample preparation, enrichment and/or depletion, separation and/or pooling. In additon as you can see above, there are a number of software processing steps. Laboratory information management systems (LIMS) can track all these operations. Proteus from Genologics is a LIMS oriented toward proteomics and mass spectrometry.
  • Compute pI/MW: This tool computes the theoretical pI (isoelectric point) and Mw (molecular weight) for a list of Swiss-Prot proteins or for a sequence of amino acids. You can use this to check that the identified protein is consistent with its position on a 2D PAGE gel. Remember that real proteins in samples are often modified in ways that change pI and Mw from these theoretical values.

Discussion Boards

  • ABRF Forum: The Association of Biological Resource Facilities (ABRF) discussion forum deals with biological techniques and proteomics methodology.
  • BioStar: Questions and answers on bioinformatics, computational genomics and systems biology.
  • Science Advisory Board: The Science Advisory Board hosts an active list with discussing a wide variety of topics, including sections on proteomics, bioinformatics, software, and protocols.
  • Chromatography Forum: Chromatography Forum is a public discussion group where you can post questions, news, or messages of interest to chromatographers everywhere.

Proteomics Database Resources

  • Proteomic and Genomic Web Services: Nucleic Acids Research also publishes an issue on web servers listing web tools that you can use such as BLAST, ESLpred, SDPpred, iMolTalk, ProMoST, and many, many others.
  • Peptide Atlas: Peptide atlas is a collection of MS/MS data that has been analyzed for peptide identification. The Open Proteomics Database, the Sashimi data repository, and Quartz also have proteomics mass spec data. The Global Proteome Machine (GPM) organization has a database for storing the results of searches by X!Tandem. The World 2D PAGE site lists 2D gels for many tissue samples.
  • HUPO - Human Proteomics Initiative: This site's goal is to annotate all the known human proteins. This annotation will include function, domain structure, subcellular location, post-translational modifications, variants, and similarities to other proteins.
  • Human Genome Variation Society: This site is your guide to databases that deal with mutations, polymorphisms, alleles, and SNPs. Some of these databases are oriented to specific human disorders. The Alternative Splicing Gallery shows how each gene is assembled into multiple proteins by splice variants.
  • ELM - Functional sites: ELM predicts functional sites in eukaryotic proteins. This site has links to dozens of other sites that examine protein motifs and domains.
  • Match Miner: MatchMiner is a set of tools that enables the user to translate between disparate ids for the same gene. It uses data from the UCSC, LocusLink, Unigene, OMIM, Affymetrix and Jackson data sources to determine how different ids relate. Supported id types include, gene symbols and names, IMAGE and FISH clones, GenBank accession numbers and UniGene cluster ids.

Tutorials and Information

  • Proteomics Interest Group at NIH: At this site you can hear the experts speak (turn on your PC's speakers) and see the slides that they presented to the NIH proteome research community. The archive has past seminars, symposiums, tutorials and lectures.
  • Isotopes and Mass Spectrometry: Atoms of carbon have isotopes of mass 12, 13, and 14 amu. Since proteins are made of carbon (and other atoms), the mass detected by the mass spectrometer for a protein depends upon the isotopes of the protein's atoms. You can learn what this means for protein identification in this IonSource tutorial. You can also develop a feel for isotopes by using Isotopica, Isotopident or an isotope calculator to work out examples.
  • Electrospray Ionization: This site is an introduction to electrospray (ESI) by a company that makes electrosprays.
  • MS Quantitation: Suppose you know what protein is in your sample. How can you measure how much of it there is? One method of measuring the amount of the protein uses a mass spectrometer and an internal standard. This tutorial from IonSource is aimed at mass spectrometrists. If you decide to do this, you can find peptides to use as internal standards at the Peptide Resource Page.
  • Antibodies tutorial: Antibodies are the gold standard in protein identification. An antibody vendor, Chemicon, has made a very nice 36 page pdf "Introduction to Antibodies". To make sense of antibody discussions, remember that what you call a protein, they call an antigen. If you want to find an antibody for a particular purpose, the Antibody Resource Page is a good place to start looking.
  • Protein chemist tutorials: This site has a number of tutorials on protein chemistry including purification, gels, antibodies, gels and modifications. It also has calculators for buffers and peptide characteristics.


  • Proteomics Glossary: This site defines many of the common terms in proteomics. It is one of a number of interrelated glossaries and many of the terms are linked.
  • Little Encyclopedia of Mass Spectrometry: Mass spectrometry has a sea of acronyms. To decode "ESI", "MALDI" and other mass spec terms, look at this cleverly constructed web page.
  • Language of HPLC: If you don’t know what HPLC means, this is the web site to look it up. By the way, HPLC means High Pressure Liquid Chromatography and it is one standard way of separating peptides that are being fed into a mass spectrometer. Like other technologies, it has its special vocabulary.
  • Biochemistry Dictionary: This site tries to help you if you are faced with “the daunting task” of acquiring the vocabulary of biochemistry. This site is not fancy, but if you are feeling daunted this might be what you need.

Academic Publications

  • Molecular & Cellular Proteomics: Most scientific journals are prohibitively expensive unless you work for an organization that subscribes to them for you. Not so with this journal which embraces open access. The papers are available free on-line from the day that they are accepted for publication. The "Papers in Press" have not yet formatted for publication.
  • Proteome Science: Proteome Science which has free on-line articles on proteomics research and methodology. There is about one new article per month. Other free journals are BMC BioTechnology and BMC BioInformatics and Nucleic Acids Research.


  • Proteomics at spectroscopyNOW: The proteomics page of spectroscopyNOW has feature articles (you need to register), readable digests of scientific papers that have popular appeal, news about proteomics uses and the proteomics industry. Another page in this site is "BASE PEAK" claims to be "the web's leading mass spectrometry resource". SeparationsNOW is another web site by the same organization that also has a section on proteomics.
  • This site is subtitled "The world of mass spectroscopy". It has news stories, interviews, product announcements and upcoming events related to mass spectrometry. About two or three new items are posted each week.
  • LCGC magazine: The tagline for this web site is "Solutions for separation scientists". This magazine is targetted toward people who do chromatography, including HPLC.
  • Biocompare: The buyer's guide for life scientists. If you want to buy a protein extraction kit and don't know where to look, this site will give you a lead.