Publications

Adaikkalam Vellaichamy, John C. Tran, Adam D. Catherman, Ji Eun Lee, John F. Kellie, Steve M. M. Sweet, Leonid Zamdborg, Paul M. Thomas, Dorothy R. Ahlf, Kenneth R. Durbin, Gary A. Valaskovic and Neil L. Kelleher

Anal. Chem., Article ASAP. Publication Date (Web): January 14, 2010

Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC−MS to drive high-throughput topdown proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle−skimmer dissociation and detection of fragment ions with <10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10−60 proteins identified in single LC−MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.

Lee JE, Kellie JF, Tran JC, Tipton JD, Catherman AD, Thomas HM, Ahlf DR, Durbin KR, Vellaichamy A, Ntai I, Marshall AG, Kelleher NL.

J Am Soc Mass Spectrom. 2009 Dec;20(12):2183-91.

For fractionation of intact proteins by molecular weight (MW), a sharply improved twodimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For topdown tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.

Witkowski C, Harkins J.

J Vis Exp. 2009 Dec 3;(34). pii: 1842. doi: 10.3791/1842.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.

John C. Tran and Alan A. Doucette

Anal. Chem., 2009, 81 (15), pp 6201–6209

Reliable size-based protein separation is an invaluable biological technique. Unfortunately, size separation in solution is underutilized, owing perhaps to the poor resolution of conventional techniques. Here, we report an enhanced multiplexed GELFrEE (gel-eluted liquid fraction entrapment electrophoresis) device which incorporates eight independent separation channels, operating with high repeatability. This enables simultaneous size separation of independent proteome samples, each into 16 well resolved liquid fractions, covering 10−150 kDa in 1.5 h. A novel strategy to increase sample loads while maintaining electrophoretic resolution is presented by distributing the sample among the eight channels with subsequent pooling of collected fractions. Liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis of the S. cerevisiae proteome following GELFrEE separation and sodium dodecyl sulfate (SDS) removal demonstrates the resolution and high correlation achieved between molecular weight and fraction number for the identified proteins. This device is highly orthogonal to solution isoelectric focusing, enabling our disclosure of a fully multiplexed high-throughput two-dimensional liquid electrophoretic (2D LE) platform that separates analogously to 2D polyacrylamide gel electrophoresis (PAGE). With 2D LE, a total of 128 well-resolved liquid fractions are obtained from 1 mg of S. cerevisiae proteins covering ranges 3.8 < pI < 7.8 and 10 kDa < MW < 150 kDa in an unprecedented 3.25 h total separation time.

John C. Tran and Alan A. Doucette

Anal. Chem., 2008, 80 (5), pp 1568–1573

Although well-established as a technique for protein purification, the application of continuous elution tube gel electrophoresis to proteome fractionation remains problematic. Difficulties associated with sample collection, particularly at the high mass range or at low sample loadings, continue to plague the technique. Furthermore, an upper mass limit is imposed as slow-moving higher molecular weight proteins are progressively diluted during the collection phase. In short, with current technology, effective separation over a broad mass range has not been achieved. In this work, we present improved techniques for continuous elution tube gel electrophoresis to accommodate broad mass range separation of proteins. Our device enables rapid partitioning of a proteome into discrete mass range fractions in the solution phase. High recovery is achieved at submicrogram to milligram sample loadings. We demonstrate comprehensive, reproducible separations of protein mixtures, as well as separation of a proteome in as fast as 1 h, over mass ranges from below 10 to 250 kDa. Finally, we identified proteins from a prefractionated standard protein mixture using liquid chromatography tandem mass spectrometric (LC−MS/MS) analysis.

Hans-Rudolf Aerni, Dale S. Cornett and Richard M. Caprioli

Anal. Chem., 2009, 81 (17), pp 7490–7495

Analysis of formalin-fixed paraffin-embedded tissues (FFPE) is increasingly recognized as a strategy for the discovery and validation of clinically useful biomarker candidates. Large tissue collections including tissue microarrays (TMAs) are available, but current analytical strategies for their characterization have limited throughput. In this report, we describe a workflow for rapid analysis of hundreds of FFPE tissue specimens. The strategy combines parallel sample processing and on-chip electrophoresis with automated matrix-assisted laser desorption ionzation mass spectrometry (MALDI MS) analysis. The method is optimized for small quantities of clinically valuable tissues allowing detection of hundreds of peptides from a single core in a TMA section. We describe results from the optimization of the method and apply it for the analysis of tissue microarrays containing formalin fixed tissue specimens from human kidney.

James B. Harkins, IV, Benjamin B. Katz, Salvador J. Pastor, Peter Osucha, Dean G. Hafeman, Charles E. Witkowski, II, and Jeremy L. Norris

Anal. Chem., 2008, 80 (8), pp 2734–2743

The preparation of complex biological samples for high-throughput mass spectrometric analyses remains a significant bottleneck, limiting advancement of the capabilities of mass spectrometry (MS) and ultimately limiting development of novel clinical assays. The removal of interfering species (e.g., salts, detergents, and buffers), concentration of dilute analytes, and the reduction of sample complexity are required in order to maximize the quality of resultant MS data. This study describes a novel sample preparation method that makes use of electrophoresis to prepare complex biological samples for high-throughput MS analysis. The method provides for integration of key sample preparation steps, including depletion, fractionation, desalting, and concentration. The prepared samples are captured onto a monolithic reversed-phase capture target that can be analyzed directly by a mass spectrometer. Up to 96 individual samples are simultaneously prepared for MS analysis in under 1 h. For standard proteins added to serum, this method provides femtomole level sensitivity and reproducible label-free detection (coefficient of variation <30%). This study demonstrates that this electrophoretic sample preparation system permits high-throughput sample preparation for mass spectrometric analysis of complex biological samples, such as serum, plasma, and tissue extracts.

Waanders LF, Chwalek K, Monetti M, Kumar C, Lammert E, Mann M.

Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):18902-7.

Technological developments make mass spectrometry (MS)-based proteomics a central pillar of biochemical research. MS has been very successful in cell culture systems, where sample amounts are not limiting. To extend its capabilities to extremely small, physiologically distinct cell types isolated from tissue, we developed a high sensitivity chromatographic system that measures nanogram protein mixtures for 8 h with very high resolution. This technology is based on splitting gradient effluents into a capture capillary and provides an inherent technical replicate. In a single analysis, this allowed us to characterize kidney glomeruli isolated by laser capture microdissection to a depth of more than 2,400 proteins. From pooled pancreatic islets of Langerhans, another type of "miniorgan," we obtained an indepth proteome of 6,873 proteins, many of them involved in diabetes. We quantitatively compared the proteome of single islets, containing 2,000-4,000 cells, treated with high or low glucose levels, and covered most of the characteristic functions of beta cells. Our ultrasensitive analysis recapitulated known hyperglycemic changes but we also find components up-regulated such as the mitochondrial stress regulator Park7. Direct proteomic analysis of functionally distinct cellular structures opens up perspectives in physiology and pathology.

Morris K, Lorenzen MD, Hiromasa Y, Tomich JM, Oppert C, Elpidina EN, Vinokurov K, Jurat-Fuentes JL, Fabrick J, Oppert B.

J Proteome Res. 2009 Aug;8(8):3889-98.

Tribolium castaneum is an important agricultural pest and an advanced genetic model for coleopteran insects. We have taken advantage of the recently acquired T. castaneum genome to identify T. castaneum genes and proteins in one of the more critical environmental interfaces of the insect, the larval alimentary tract. Genetic transcripts isolated from the T. castaneum larval gut were labeled and hybridized to a custom array containing oligonucleotides from predicted genes in the T. castaneum genome. Through a ranking procedure based on relative labeling intensity, we found that approximately 17.6% of the genes represented in the array were predicted to be highly expressed in gut tissue. Several genes were selected to compare relative expression levels in larval gut, head, or carcass tissues using quantitative real-time PCR, and expression levels were, with few exceptions, consistent with the gut rankings. In parallel with the microarrays, proteins extracted from the T. castaneum larval gut were subjected to proteomic analysis. Two-dimensional electrophoretic analysis combined with MALDI-TOF resulted in the identification of 37 of 88 selected protein samples. As an alternative strategy, one-dimensional electrophoretic separation of T. castaneum larval gut proteins followed by two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of 98 proteins. A comparison of the proteomic studies indicated that 16 proteins were commonly identified in both, whereas 80 proteins from the proteomic analyses corresponded to genes with gut rankings indicative of high expression in the microarray analysis. These data serve as a resource of T. castaneum transcripts and proteins in the larval gut and provide the basis for comparative transcriptomic and proteomic studies related to the gut of coleopteran insects.

H. Mikkelsen, N. J. Bond, M. E. Skindersoe, M. Givskov, K. S. Lilley and M. Welch

Microbiology 155 (2009), 687-698; DOI 10.1099/mic.0.025551-0

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals. It is also a model organism for bacterial biofilm formation. Acute infections are often associated with planktonic or freefloating cells, high virulence and fast growth. Conversely, chronic infections are often associated with the biofilm mode of growth, low virulence and slow growth that resembles that of planktonic cells in stationary phase. Biofilm formation and type III secretion have been shown to be reciprocally regulated, and it has been suggested that factors related to acute infection may be incompatible with biofilm formation. In a previous proteomic study of the interrelationships between planktonic cells, colonies and continuously grown biofilms, we showed that biofilms under the growth conditions applied are more similar to planktonic cells in exponential phase than to those in stationary phase. In the current study, we investigated how these conditions influence the production of virulence factors using a transcriptomic approach. Our results show that biofilms express the type III secretion system, whereas planktonic cells do not. This was confirmed by the detection of PcrV in the cellular and secreted fractions of biofilms, but not in those of planktonic cells. We also detected the type III effector proteins ExoS and ExoT in the biofilm effluent, but not in the supernatants of planktonic cells. Biofilm formation and type III secretion are therefore not mutually exclusive in P. aeruginosa, and biofilms could play a more active role in virulence than previously thought.

deWilde A, Sadilkova K, Sadilek M, Vasta V, Hahn SH.

Clin Chem. 2008 Dec;54(12):1941-2.

Newborn screening to identify infants with treatable congenital disorders is carried out worldwide. Recent tandem mass spectrometry (MS/MS) applications have markedly expanded the ability to screen for >50 metabolic diseases with a single dried blood spot (DBS). The feature that makes metabolic disorders particularly amenable to screening is the presence of specific small-molecule metabolites. Many treatable disorders such as Wilson disease, however, are characterized by absent or diminished large proteins in plasma or within circulating blood cells, for which there are currently no cost-effective screening methods. METHODS: We developed an assay for quantifying ceruloplasmin (CP) in DBS for newborn screening of Wilson disease. CP-specific peptides from DBS samples digested by trypsin were quantified using isotopically labeled peptide internal standards and liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). RESULTS: The calibration curve was linear from 20 to 95 mg/dL (200-950 mg/L). Intraassay imprecision (mean CV) for CP concentrations of 25, 35, and 55 mg/dL (250, 350, and 550 mg/L) was 9.2%, 10.7%, and 10.2%, respectively. Interassay imprecision for 19 different batches was 8.9%, 5.8%, and 6.9%. A method comparison study on previously tested patient samples for CP gave comparable results with lower limit of quantification, around 0.7 mg/dL (7 mg/L). CONCLUSIONS: Our study supports that newborn screening for Wilson disease is feasible using LC-MS/MS assay for CP quantification in DBS after tryptic digestion. This approach should be applicable to newborn screening for other treatable genetic conditions, such as primary immunodeficiencies, that have large proteins as biomarkers.

Beyer NH, Schou C, Houen G, Heegaard NH.

J Immunol Methods. 2008 Jan 31;330(1-2):24-33.

A method for the identification of protein antigens captured in electroimmunoprecipitates was developed. Different antigen-antibody precipitates were generated by agarose gel immunoelectrophoresis. The immunoprecipitates were excised and various methods for extracting and dissociating the precipitates were systematically studied by analyzing for protein components of the extracts using peptide mass fingerprinting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal recovery of antigen was obtained by 24-h extraction at 37 degrees C using a minimal volume of 0.06 M Tris-HCl, 10% SDS (pH 7). This simple and robust method is useful for the characterization of antibody specificity. It can also be used to identify antigens generating unknown precipitates in crossed immunoelectrophoresis with polyspecific antisera, including human IgG-antigen complexes electroimmunoprecipitated by secondary antibodies. Thus, the method may prove useful as an additional technique in biomarker discovery.

Chen EI, Cociorva D, Norris JL, Yates JR 3rd.

J Proteome Res. 2007 Jul;6(7):2529-38. Epub 2007 May 27.

An optimization and comparison of trypsin digestion strategies for peptide/protein identifications by microLC-MS/MS with or without MS compatible detergents in mixed organic-aqueous and aqueous systems was carried out in this study. We determine that adding MS-compatible detergents to proteolytic digestion protocols dramatically increases peptide and protein identifications in complex protein mixtures by shotgun proteomics. Protein solubilization and proteolytic efficiency are increased by including MS-compatible detergents in trypsin digestion buffers. A modified trypsin digestion protocol incorporating the MS compatible detergents consistently identifies over 300 proteins from 5 microg of pancreatic cell lysates and generates a greater number of peptide identifications than trypsin digestion with urea when using LC-MS/MS. Furthermore, over 700 proteins were identified by merging protein identifications from trypsin digestion with three different MS-compatible detergents. We also observe that the use of mixed aqueous and organic solvent systems can influence protein identifications in combinations with different MS-compatible detergents. Peptide mixtures generated from different MS-compatible detergents and buffer combinations show a significant difference in hydrophobicity. Our results show that protein digestion schemes incorporating MS-compatible detergents generate quantitative as well as qualitative changes in observed peptide identifications, which lead to increased protein identifications overall and potentially increased identification of low-abundance proteins.