Analytical techniques for cell fractions

Anderson, Norman G.; Willis, D.D.; Holladay, D.W.; Caton, J.E.; Holleman, J.W.; Eveleigh, J. W.; Attrill, J.E.; Ball, Frances L.; Anderson, N. Leigh

doi:10.1016/0003-2697(75)90634-x

Cited by 24 publications

(5 citation statements)

References 5 publications

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“…Such an enrichment method can be applied routinely if the antibody adsorbent is capable of being recycled, a property which has been well-established, at least for polyclonal antibodies and protein ligands. 8,13,14 We have investigated an approach (summarized in Figure 1) that we refer to by the acronym SISCAPA (Stable Isotope Standards with Capture by Anti-Peptide Antibodies). SISCAPA combines the four elements previously described: (1) digestion of a protein sample to peptides; (2) addition of internal standard peptides labeled with stable isotopes; (3) enrichment of low abundance peptides by capture with immobilized antibodies; and (4) quantitation by MS.…”

Section: Introductionmentioning

confidence: 99%

“…An attractive alternative is the use of specific antibody affinity reagents to capture the desired peptides followed by their quick elution into the mass spectrometer. Such an enrichment method can be applied routinely if the antibody adsorbent is capable of being recycled, a property which has been well-established, at least for polyclonal antibodies and protein ligands. ,, …”

Section: Introductionmentioning

confidence: 99%

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Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

et al. 2004

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A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

et al. 2004

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“…In addition, when antibody amount is limiting, the ability to carry out multiple successive captures from a large pool of antigen allows accumulation of much more antigen than the available amount of antibody. 11 Polyclonal antibody columns have been shown to be recyclable, some hundreds of times, 12 but not all antibodies survive the harsh elution conditions. This fact makes it important to select antibodies that can withstand multiple loadelution cycles.…”

Section: Discussionmentioning

confidence: 99%

“…Although antibodies are not often reused in most current ELISA or immunoprecipitation protocols, there are important advantages to re-usable antibodies for online enrichment or subtraction, not the least of which is lower cost per cycle. In addition, when antibody amount is limiting, the ability to carry out multiple successive captures from a large pool of antigen allows accumulation of much more antigen than the available amount of antibody . Polyclonal antibody columns have been shown to be recyclable, some hundreds of times, but not all antibodies survive the harsh elution conditions.…”

Section: Discussionmentioning

confidence: 99%

An Effective and Rapid Method for Functional Characterization of Immunoadsorbents Using POROS Beads and Flow Cytometry

Anderson¹,

Haines²,

Tw³

2004

J. Proteome Res.

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To facilitate the construction, functional characterization, and use of immunoadsorbents, we have developed a flow cytometry method that allows rapid assessment of large numbers of particle-bound antibodies. Protein G derivitized POROS beads were used to bind affinity-purified antibodies specific for synthetic peptides designed from human plasma proteins. The antibodies were covalently coupled to the beads and used to capture and release synthetic peptides that had been labeled at the C-terminus with the fluorochrome Alexa Fluor 488. Antibody coupling and specificity of antigen binding and release were measured by analysis of the POROS affinity beads by flow cytometry. The affinity-capture matrixes were also used through several antigen-binding and release cycles without loss of peptide binding efficiency. The ability to produce and characterize extremely small amounts of POROS affinity matrices will facilitate their use in protein microchemical procedures such as protein chip technology, monoclonal antibody screening and mass spectrometry, applications where analytes are limiting or present in low abundance in complex mixtures.

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“…The strategy was to develop several different technologies in parallel. These included some of the first high pressure liquid chromatographic systems to resolve low molecular weight compounds [6] including sugars [7] and the constituents of nucleic acids [8], high resolution centrifuges to resolve cell components [9,10], and methods to separate proteins including rapid recycling immuno-affinity chromatographic systems to simplify complex protein mixtures [11,12]. Since it was realized that automatic fractionation methods would yield large numbers of samples to be analyzed for enzyme activities, the first computerized enzyme analyzer (the centrifugal fast analyzer) was invented, developed, and came into general use in clinical chemistry laboratories worldwide [13].…”

Section: History Of Molecular Anatomymentioning

confidence: 99%

Back to the future: The human protein index (HPI) and the agenda for post-proteomic biology

2001

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The effort to produce an index of all human proteins (the human protein index, or HPI) began twenty years ago, before the initiation of the human genome program. Because DNA sequencing technology is inherently simpler and more scalable than protein analytical technology, and because the finiteness of genomes invited a spirit of rapid conquest, the notion of genome sequencing has displaced that of protein databases in the minds of most molecular biologists for the last decade. However, now that the human genome sequence is nearing completion, a major realignment is under way that brings proteins back to the center of biological thinking. Using an influx of new and improved protein technologies--from mass spectrometry to re-engineered two-dimensional (2-D) gel systems, the original objectives of the HPI have been expanded and the time frame for its execution radically shortened. Several additional large scale technology efforts flowing from the HPI are also described.

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Analytical techniques for cell fractions

Cited by 24 publications

References 5 publications

Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

An Effective and Rapid Method for Functional Characterization of Immunoadsorbents Using POROS Beads and Flow Cytometry

Back to the future: The human protein index (HPI) and the agenda for post-proteomic biology

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