Improved Electrospray Ionization Efficiency Compensates for Diminished Chromatographic Resolution and Enables Proteomics Analysis of Tyrosine Signaling in Embryonic Stem Cells

Ficarro, Scott B.; Zhang, Yi; Lu, Yu; Moghimi, Ahmadali R.; Askenazi, Manor; Hyatt, Elzbieta; Smith, Eric; Boyer, Leah; Schlaeger, Thorsten M.; Luckey, Chance John; Marto, Jarrod A.

doi:10.1021/ac802720e

Cited by 101 publications

(138 citation statements)

References 66 publications

(81 reference statements)

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“…Already, the method described here could be used to evaluate single islets developed from in vitro differentiated stem cells or disease control of a few islets taken from human biopsies. The flow rate and column diameter can be significantly lowered (37) and MS sensitivity and scan speed increased (38). Thus, a few hundred cells are analyzable or alternatively depth of coverage can be increased significantly.…”

Section: Resultsmentioning

confidence: 99%

Quantitative proteomic analysis of single pancreatic islets

Waanders

Chwalek

Monetti

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

200

213

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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 in-depth 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.hyperglycemia ͉ liquid chromatography-mass spectrometry ͉ quantitative proteomics ͉ replay technology

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Section: Resultsmentioning

confidence: 99%

Quantitative proteomic analysis of single pancreatic islets

Waanders

Chwalek

Monetti

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

200

213

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“…The reverse phase C 18 was performed using an in-house C 18 capillary column packed with 5-m C 18 Magic bead resin (Michrom; 75-m inner diameter and 12-cm bed length) on an 1100 Agilent HPLC system (25). The mobile phase buffer consisted of 0.1% HCOOH in ultrapure water with the eluting buffer of 100% CH 3 CN run over a shallow linear gradient over 60 min with a flow rate of 0.3 l/min.…”

Section: Mass Spectrometry Analysesmentioning

confidence: 99%

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Iliuk

Martin

Alicie

et al. 2010

Molecular & Cellular Proteomics

153

180

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The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro-and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. Molecular & Cellular Proteomics 9:2162-2172, 2010.Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple cellular processes (1, 2). Mass spectrometry-based phosphoproteomics provides a method for the global analysis of protein phosphorylation and molecular signaling in cells (3,4). Despite the great progress that has been made over the past few years, the isolation of phosphopeptides and their analysis by mass spectrometry are still a considerable challenge because of the typically low stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. An early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably with high efficiency, selectivity, sensitivity, and reproducibility. Currently, there are three major strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5-8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture techniques use immobilized metal affinity chromatography (IMAC) with Fe(III) (11,12) or Ga(III) (13) and, for the past a few years, more successful metal oxide approaches (i.e. TiO 2 (14, 15) and ZrO 2 (16, 17)) for the selective binding of phosphorylated peptides. Almost all of the current isolation methods are b...

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“…Precolumns with cast chemical frits were slurry-packed to 5 cm in length with a stationary phase consisting of a 5-m diameter, 100 Å pore size, C18 particles (Magic C18AQ, Michrom Bioresources, Inc., Auburn CA) (46). Reversed-phase LC separation was achieved across a 13-cmlong, 50-m inner diameter ϫ 360-m outer diameter fusedsilica analytical column packed with the same C18 stationary phase.…”

Section: Liquid Chromatography Electrospray Tandem Mass Spectrometry mentioning

confidence: 99%

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Yang

Park

Davis

et al. 2012

Journal of Biological Chemistry

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Background: ␣1(V) is an extensively modified collagen chain important in disease. Results: Comprehensive mapping of ␣1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets. Conclusion:The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties. Significance: Positions, numbers, and occupancy of modified sites can provide insights into ␣1(V) biological properties.

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Improved Electrospray Ionization Efficiency Compensates for Diminished Chromatographic Resolution and Enables Proteomics Analysis of Tyrosine Signaling in Embryonic Stem Cells

Cited by 101 publications

References 66 publications

Quantitative proteomic analysis of single pancreatic islets

Quantitative proteomic analysis of single pancreatic islets

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

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