Methods for fractionation, separation and profiling of proteins and peptides

Issaq, Haleem J.; Conrads, Thomas P.; Janini, George M.; Veenstra, Timothy D.

doi:10.1002/1522-2683(200209)23:17<3048::aid-elps3048>3.0.co;2-l

Cited by 203 publications

(145 citation statements)

References 84 publications

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“…Several fractionation techniques have been developed to separate and consequently quantify the proteins in serum (Issaq et al 2002). Most of them depend on the initial determination of total serum proteins, and then the concentrations of the main fractions are calculated according to the used method.…”

Section: Albumin and Globulinsmentioning

confidence: 99%

Serum proteins and their diagnostic utility in veterinary medicine: a review

Tóthová¹,

Nagy²,

Kováč³

2016

Vet. Med.

134

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ABSTRACT:The evaluation of serum proteins and their electrophoretic pattern is a well-established laboratory method in the diagnosis of many diseases in humans, which has replaced the biochemical determination of the concentrations of albumin and the ratio of albumin to globulins. The measurement of serum proteins may be an important diagnostic tool for the detection, diagnosis, and monitoring of various diseases and pathological processes. The results of serum protein electrophoresis can be one of the most useful diagnostic aids in a wide spectrum of diseases, including infectious and inflammatory diseases, renal or gastrointestinal disorders, immunodeficiency states, as well as paraproteinaemias caused by lymphoid or plasma cell neoplasia. Although many studies have been carried out to determine the usefulness of the determination of serum proteins and their electrophoretic pattern in various disease conditions and disorders also in animals, serum protein evaluation is still a relatively little-used diagnostic tool in veterinary medicine. In this article, methods of serum protein determination, their diagnostic utility in animal care practice and their different patterns in dysproteinaemias and paraproteinaemias are reviewed.

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Section: Albumin and Globulinsmentioning

confidence: 99%

Serum proteins and their diagnostic utility in veterinary medicine: a review

Tóthová¹,

Nagy²,

Kováč³

2016

Vet. Med.

134

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“…Not only are certain classes of proteins underrepresented by 2D-PAGE, the subsequent identification of the separated proteins is laborious and time-consuming (3). Alternative approaches for the identification of proteins circumvent the need for 2D gel fractionation and rely solely on multidimensional chromatographic separation of proteolytically digested proteins prior to MS/MS analysis (1,4). While these solution-based methods do not provide a direct method for protein quantitation, they routinely are capable of providing well over 1,000 protein identifications in rapid (i.e.…”

Section: Molecular and Cellular Proteomics 3:896 -907 2004mentioning

confidence: 99%

Global Analysis of the Cortical Neuron Proteome

Conrads

et al. 2004

Molecular & Cellular Proteomics

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In this study, a multidimensional fractionation approach was combined with MS/MS to increase the capability of characterizing complex protein profiles of mammalian neuronal cells. Proteins extracted from primary cultures of cortical neurons were digested with trypsin followed by fractionation using strong cation exchange chromatography. Each of these fractions was analyzed by microcapillary reversed-phase LC-MS/MS. The analysis of the MS/MS data resulted in the identification of over 15,000 unique peptides from which 3,590 unique proteins were identified based on protein-specific peptide tags that are unique to a single protein in the searched database. In addition, 952 protein clusters were identified using cluster analysis of the proteins identified by the peptides not unique to a single protein. This identification revealed that a minimum of 4,542 proteins could be identified from this experiment, representing ϳ16% of all known mouse proteins. An evaluation of the number of false-positive identifications was undertaken by searching the entire MS/MS dataset against a database containing the sequences of over 12,000 proteins from archaea. This analysis allowed a systematic determination of the level of confidence in the identification of peptides as a function of SEQUEST cross correlation (X corr ) and delta correlation (⌬Cn) scores. Correlation charts were also constructed to show the number of unique peptides identified for proteins from specific classes. The results show that low-abundance proteins involved in signal transduction and transcription are generally identified by fewer peptides than high-abundance proteins that play a role in maintaining mammalian cellular structure and motility. The results presented here provide the broadest proteome coverage for a mammalian cell to date and show that MS-based proteomics has the potential to provide high coverage of the proteins expressed within a cell.

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“…The protein complexity of biological samples may be simplified through fractionation based on different protein properties, including sequential solubilization, selective precipitation, and affinity purification, or through various chromatography-based PROTEOME ANALYSIS WITH MASS SPECTROMETRY & methods (Issaq et al, 2002;Liu et al, 2002;.…”

Section: Fractionation Of Proteins Based On Their Chemical and Phmentioning

confidence: 99%

Intact‐protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

Wang

Hanash

2004

Mass Spectrometry Reviews

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The complexity of tissue and cell proteomes and the vast dynamic range of protein abundance present a formidable challenge for analysis that no one analytical technique can overcome. As a result, there is a need to integrate technologies to achieve the high-resolution and high-sensitivity analysis of complex biological samples. The combined technologies of separation science and biological mass spectrometry (Bio-MS) are the current workhorse in proteomics, and are continuing to evolve to meet the needs for high sensitivity and high throughput. They are relied upon for protein quantification, identification, and analysis of post-translational modifications (PTMs). The standard technique of two dimensional poly-acrylamide gel electrophoresis (2D PAGE) offers relatively limited resolution and sensitivity for the simultaneous analysis of all cellular proteins, with only the most highly abundant proteins detectable in whole cell or tissuederived samples. Hence, many alternative strategies are being explored. Numerous sample preparation procedures are currently available to reduce sample complexity and to increase the detectability of low-abundance proteins. Maintaining proteins intact during sample preparation has important advantages compared with strategies that digest proteins at an early step. These strategies include the ability to quantitate and recover proteins, and the assessment of PTMs. A review of current intact protein-based strategies for protein sample preparation prior to mass spectrometry (MS) is presented in the context of biomedically driven applications. # 2004 Wiley Periodicals, Inc., Mass Spec Rev 24: [413][414][415][416][417][418][419][420][421][422][423][424][425][426] 2005

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Methods for fractionation, separation and profiling of proteins and peptides

Cited by 203 publications

References 84 publications

Serum proteins and their diagnostic utility in veterinary medicine: a review

Serum proteins and their diagnostic utility in veterinary medicine: a review

Global Analysis of the Cortical Neuron Proteome

Intact‐protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

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