Integrating “Top-Down” and “Bottom-Up” Mass Spectrometric Approaches for Proteomic Analysis of Shewanella oneidensis

Abstract

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.

Characterization of secreted proteins of 2-cell mouse embryos cultured in vitro to the blastocyst stage with and without protein supplementation

“…The generated peptides were extracted with 50 % acetonitrile/0.1 % formic acid, and dried before reconstituting in 0.1 % formic acid. The peptides were separated on a C18 column (ResPrep C18 column, Bellefonte, PA, USA) and subjected to nano high performance liquid chromatography (HPLC, Accella, Thermo Fisher) and electro spray ionization (ESI) tandem mass spectrometry (LC-ESI-MS-MS) with a linear trap quadrupole instrument (LTQ, Thermo Fisher Scientific, Waltham, MA) [10][11][12][13]. Tryptic peptides extracted from identical sectors of the same media categories were combined to increase the concentration of the peptides 3-fold prior to mass spectrometry on the LTQ.…”

Capillary separations enabling tissue proteomics‐based biomarker discovery

“…Total peak capacity improvements in multidimensional chromatography platforms have increased the number of detected peptides and proteins identified due to better use of the MS dynamic range and reduced discrimination during ionization. To increase the proteome coverage, particularly for the identification of low-abundance proteins, these peptide-based proteome technologies often require large quantities of enzymatically/chemically cleaved peptides, ranging from a few milligrams [34,35] to several hundreds of micrograms [33,36,37], and are generally incompatible with protein levels extracted from microdissection-procured tissue samples.…”

Hydrophilic immobilized trypsin reactor with magnetic graphene oxide as support for high efficient proteome digestion

“…Protein separation by gel or liquid chromatography combined with enzymatic cleavage of the intact protein by proteases followed by mass spectrometry (MS) determination of generated peptides and subsequent database searching, has become a popular technique for bottom-up strategy based proteomic study [1][2][3][4][5][6][7]. In this peptide-based technology, rapid and complete digestion of all proteins is crucial to the throughput and accuracy of protein identification.…”