Quantitative Proteomics Using Ultralow Flow Capillary Electrophoresis–Mass Spectrometry

In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE–MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE–MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE–MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE–MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE–MS analysis

<i>In vivo</i> stability of ⁶⁸Ga-labelled mono- and multimeric Nle-MG based conjugates.

<p>Representative radio-RP-HPLC chromatograms of supernatants analysed from <i>in vivo</i> samples, serum (A), urine (B), kidney- (C) and liver homogenate (D). (IP = intact peptide).</p

Distribution coefficient and protein binding of mono- and multimeric conjugates of two different MG analogues (-Met/Nle) radiolabelled with gallium-68.

<p>Distribution coefficient and protein binding of mono- and multimeric conjugates of two different MG analogues (-Met/Nle) radiolabelled with gallium-68.</p

Receptor affinity studies (IC₅₀) with ^natGa-bound peptides as competitor and human ¹²⁵I-[Leu¹⁵]-Gastrin as radioligand on whole A431-CCK2R cells.

<p>Receptor affinity studies (IC₅₀) with ^natGa-bound peptides as competitor and human ¹²⁵I-[Leu¹⁵]-Gastrin as radioligand on whole A431-CCK2R cells.</p

<i>In vitro</i> metabolite assessment of ⁶⁸Ga-labelled trimers by artificial enzymatic degradation.

<p>Representative radio-RP-HPLC chromatograms of enzymatic degradation of ⁶⁸Ga-labelled trimeric Met- (A) and Nle (B) conjugates <i>in vitro</i> (major metabolite formation assigned with arrows).</p

Cell-uptake studies of radiopeptides on CCK2R positive cells.

<p>Internalization studies of A431-CCK2R cells incubated with ⁶⁸Ga-labelled mono- and multimeric Nle-derivatives. Blocking was performed with pentagastrin in 100-fold molar excess over the conjugate.</p

<i>In vitro</i> stability assessment of radiopeptides in rat organ homogenates.

<p>⁶⁸Ga-labelled mono- and multimeric peptide conjugates incubated with varying concentrations, 1% (A) and 10% (B) respectively, of rat kidney and liver homogenates. Data are presented as mean (n = 2), error bars omitted.</p

Synthesis of FSC-based mono- and multivalent conjugates.

<p>Schematic overview on the synthetical pathway of mono- and multimeric FSC-based minigastrin bioconjugates (stereochemistry omitted).</p

Cell-binding studies of ⁶⁸Ga-labelled trimers on CCK2-mock negative cells.

<p>Cell-uptake studies of A431-mock cells incubated with ⁶⁸Ga-trimer-Met and ⁶⁸Ga-trimer-Nle (MB = membrane bound; CB = cell bound).</p

Corresponding tumour-to-organ ratios of methionine (Met) and norleucine (Nle) containing FSC-based MG mono- and multimers radiolabelled with gallium-68, data are presented as mean ± SD.

<p>Corresponding tumour-to-organ ratios of methionine (Met) and norleucine (Nle) containing FSC-based MG mono- and multimers radiolabelled with gallium-68, data are presented as mean ± SD.</p