Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

Abstract

Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed as a means to pursue top-down sequencing for protein identification. Methods have been developed to enzymatically digest all proteins in an IEF gel simultaneously, leaving the polyacrylamide gel attached to its polyester support. By retaining all gel pieces and their placement relative to one another, sample handling and tracking are minimized, and comparison to 2-D gel images is facilitated. MALDI-MS and MS/MS can then be performed directly from dried, matrix-treated IPG strips following whole gel trypsin digestion, bottom-up methodology.Side-to-side proteomics, highlighting the link between virtual and classical 2-D gel electrophoresis, is introduced to describe a method whereby intact masses are measured from one side (the IEF gel), while proteins are identified based on analyses performed from the other side (the SDS-PAGE gel). Keywords: archaea, proteomics, virtual 2-D gel electrophoresis, immobilized pH gradient gels, Methanosarcina acetivoransAbbreviations: 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; CHAPS, 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate; DTT, dithiothreitol; HDL, high density lipoprotein; ICAT, Isotope coded affinity tagging; IEF, isoelectric focusing; IPG, immobilized pH gradient; ISD, in-source dissociation; MudPIT, multidimensional protein identification technology; PSD, post-source decay; QqTOF, quadrupole time-of-flight; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TOF, time-of-flight 3 IntroductionIntact mass measurements, in concert with protein identifications, can alert one to 1) coor post-translationally modified proteins, 2) alternative splice variants, 3) RNA editing events, or Without the link to protein identity, an intact mass has limited value. Hence, tying the intact masses recovered from virtual 2-D gels to unambiguous protein identities and further to descriptors is important. We consider 3 ways to forge this link; two are well-described in popular parlance as: "top-down" and "bottom-up" [9,10]. We call the third, "side-to-side", because it highlights the link to classical 2-D gel electrophoresis: namely that isoelectric focusing constitutes one side/dimension/axis/gel in classical 2-D analysis, while SDS-PAGE constitutes the second side/dimension/axis/gel. In side-to-side analysis, one measures the intact mass from one side (the IEF gel), while securing the protein identification from an analysis performed on the other side (the SDS-PAGE gel).Top-down proteomics strategies provide both accurate intact mass and sequence da...

Mass spectrometry for proteomics

“…These are usually conducted in two workflows. “Sort-then-break” approaches are performed using off-line protein fractionation and separation prior to protein digestion, followed by direct peptide analysis by “peptide mass fingerprinting” (PMF) [26] or further peptide separation by LC interfaced to a tandem mass spectrometer [27]. In “break-then-sort” approaches, protein digestion is performed without any pre-fractionation/separation and peptides are separated by multi-dimensional chromatography followed by tandem mass spectrometric analysis, typically using rapidly scanning analyzers such as IT mass spectrometers.…”

Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem–loop-binding proteins that contributes to high-affinity RNA binding

“…This can be done by bottom-up approaches, often by using isotopically labeled peptides as internal standards (16). With recent improvements in Fourier transform ion cyclotron resonance (FTICR) MS technology and the development of new MS dissociation techniques, top-down proteomics offers a practical alternative (17)(18)(19)(20)(21)(22). In top-down experiments, sequence information is obtained from analysis of the intact protein, and quantitative information about the stoichiometry and proportion of modifications can be obtained.…”

Proteome imaging: A closer look at life's organization

“…Although the information density in their CIEF-FTICRMS study was much greater, the use of image representation permits an easy comparison between the two approaches. Orgozalek-Loo et al have employed a similar visualization strategy to reconstruct a virtual 2-D gel, using data collected from each excised gel band that was analyzed using a protocol combining gel excision, in-gel digestion, reduction, alkylation and trypsin digestion followed by MALDI-MS [18]. To deal with this wealth of data various bioinformatics initiatives have been started aimed at finding novel ways to process and extract information from data and combine different information streams.…”