Quantitative study of the interactome of PKCζ involved in the EGF-induced tumor cell chemotaxis.

Chemotaxis plays an important role in metastasis. In our previous studies, we reported that protein kinase C ζ (PKCζ) mediated cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. To further study the molecular mechanism of chemotaxis, mass spectrometry-based approaches were employed to investigate the interactome of PKCζ and its changes upon stimulation by epidermal growth factor (EGF). As a result, 233 proteins were identified as potential PKCζ binding partners. Label free quantification was applied to examine the quantitative changes of these interactions involved in the EGF induced chemotaxis. Fifteen identified proteins were enriched and 9 proteins were reduced in the presence of EGF (≥ 1.5 folds, p ≤ 0.05). The interaction between cofilin-1 (CFL1) and PKCζ was evidenced and this interaction was enhanced in the EGF induced chemotaxis signaling transduction. In addition, novel PKCζ interacting proteins potentially related with chemotaxis were characterized, such as isoform 1 of nucleophosmin (NPM1). Furthermore, Western blotting and chemotaxis assays were also applied to validate the proteomics result and explore its biological implications. Collectively, the combination of quantitative proteomics and biological assays provides a powerful strategy for elucidating the signaling pathway of tumor cell chemotaxis.

A comprehensive and non-prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine.

Increasing attention has been paid to the urinary proteome because it holds the promise of discovering various disease biomarkers. However, most of the urine proteomics studies routinely relied on protein pre-fractionation and so far did not present characterization on phosphorylation status. Two robust approaches, integrated multidimensional liquid chromatography (IMDL) and Yin-yang multidimensional liquid chromatography (MDLC) tandem mass spectrometry, were recently developed in our laboratory, with high-coverage identification of peptide mixtures. In this study, we adopted a strategy without pre-fractionation on the protein level for urinary proteome identification, using both the IMDL and the Yin-yang MDLC methods for peptide fractionation followed by identification using a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer with high resolution and mass accuracy. A total of 1310 non-redundant proteins were highly confidently identified from two experiments, significantly including 59 phosphorylation sites. More than half the annotated identifications were membrane-related proteins. In addition, the lysosomal as well as kidney-associated proteins were detected. Compared with the six largest datasets of urinary proteins published previously, we found our data included most of the reported proteins. Our study developed a robust approach for exploring the human urinary proteome, which would provide a catalogue of urine proteins on a global scale. It is the first report, to our best knowledge, to profile the urinary phosphoproteome. This work significantly extends current comprehension of urinary protein modification and its potential biological significance. Moreover, the strategy could further serve as a reference for biomarker discovery.

Fully automatic separation and identification of phosphopeptides by continuous pH-gradient anion exchange online coupled with reversed-phase liquid chromatography mass spectrometry.

Most current technologies for the enrichment of phosphopeptides rely on a tandem combination of different chromatography modes. Here, a fully automatic two-dimensional liquid chromatography mass spectrometry method was developed for global phosphopeptide identification. The peptide mixtures were loaded on a strong anion exchange (SAX) column under basic pH conditions and eluted with a continuous gradient to pH 2.0. This SAX system could be coupled online with reversed-phase liquid chromatography mass spectrometry (RP-LC-MS/MS). For peptide digests from a standard protein mixture spiked with synthesized phosphopeptides, most of the nonphosphorylated peptides were eluted in more basic pH than phosphopeptides, and the phosphopeptides were focused to acidic pH ranges and gradually eluted according to the phosphorylated states of peptides. Compared with the pH step elution method, the continuous gradient method displayed better repeatability and less peptide cross-overlap between fractions. This system provided a robust and fully automatic approach to large-scale phosphoproteomic profiling. For protein tryptic digests from HeLa cells, 1833 nonredundant phosphorylation sites were identified based on this two-phase separation. Compared with the method combining cation exchange and titanium dioxide, this anion-exchange based system preferred to identify more acidic and multiphosphorylated peptides. It also covered a more complete series of phosphorylation states of peptides.

Fractionation of complex protein mixture by virtual three-dimensional liquid chromatography based on combined pH and salt steps.

The complexity and diversity of biological samples in proteomics require intensive fractionation ahead of mass spectrometry identification. This work developed a chromatographic method called virtual three-dimensional chromatography to fractionate complex protein mixtures. By alternate elution with different pHs and salt concentrations, we implemented pH and salt steps by turns on a single strong cation exchange column to fully exploit its chromatographic ability. Given standard proteins that were not resolved solely by pH or salt gradient elution could be successfully separated using this combined mode. With a reversed phase column tandem connected behind, we further fractionated as well as desalted proteins as the third dimension. This present strategy could readily be adapted with respect to special complexity of biological samples. Crude plasma without depleting high abundance proteins were fractionated by this three-dimensional mode and then analyzed by reversed phase liquid chromatography coupled with LTQ mass spectrometry. In total, 1933 protein groups with wide dynamic ranges were identified from a single experiment. Some characteristics that correlated to the behavior of proteins on strong cation exchange columns are also discussed.

A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysis.

The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome.

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry.

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.

Proteomic analysis with integrated multiple dimensional liquid chromatography/mass spectrometry based on elution of ion exchange column using pH steps.

A novel integrated multidimensional liquid chromatography (IMDL) method is demonstrated for the separation of peptide mixtures by two-dimensional HPLC coupled with ion trap mass spectrometry. The method uses an integrated column, containing both strong cation exchange and reversed-phase sections for two-dimensional liquid chromatography. The peptide mixture was fractionated by a pH step using a series of pH buffers, followed by reversed-phase chromatography. Since no salt was used during separation, the integrated multidimensional liquid chromatography can be directly connected to mass spectrometry for peptide analysis. The pH buffers were injected from an autosampler, and the entire process can be carried out on a one-dimensional liquid chromatography system. In a single analysis, the IMDL system, coupled with linear ion trap mass spectrometry, identified more than 2000 proteins in mouse liver. The peptides were eluted according to their pI distribution. The resolution of the pH fractionation is approximately 0.5 pH unit. The method has low overlapping across pH fractions, good resolution of peptide mixture, and good correlation of peptide pIs with pH steps. This method provides a technique for large-scale protein identification using existing one-dimensional HPLC systems.

Identification of proteins separated by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis with matrix-assisted laser desorption/ionization ion trap mass spectrometry; comparison with matrix-assisted laser desorption/ionization time-of-flight mass fingerprinting.

Digests from ten gel bands containing low abundance proteins were analyzed by both matrix-assisted laser desorption/ionization ion trap (MALDI-IT) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) methods. MALDI-TOF techniques were able to identify only one protein from all 10 gel bands, while MALDI-IT identified eight proteins from the same 10 bands. The ability to perform MS/MS experiments with a MALDI-IT instrument leads to protein identifications based on both peptide molecular mass and sequence information, and is much less prone to errors and uncertainties introduced by peptide fingerprinting methodologies in which protein identification is based on peptide molecular masses alone.