The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

Endocytosis of the growth hormone receptor (GHR) is regulated by the ubiquitin-conjugating system. A cytosolic 10 amino acid motif, referred to as the ubiquitin-dependent endocytosis (UbE) motif, is involved in the ubiquitination as well as in the endocytosis of the receptor. Proteins that are implicated in one of these processes have not been identified so far. Using a glutathione S-transferase (GST)-pulldown assay with a GST fusion protein encompassing the UbE motif of the GHR, a 35 kDa protein was purified. The protein was identified by MS as small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (SGT). We found that GHR interacts with SGT. In vivo, both the precursor and the mature form of the receptor interacted with SGT. Inactivation of the ubiquitin-conjugating system did not affect the GHR-SGT interaction. Binding studies showed that the first TPR motif of SGT interacts with the UbE motif of the GHR. Taken together, these data show that SGT is a GHR-interacting protein, which binds independent of the ubiquitin-conjugating system.

Bactericidal antibodies directed against surface loops of class 1 outer membrane proteins play a crucial role in protection against meningitis and sepsis caused by Neisseria meningitidis. So far, all efforts to obtain protective antibodies against these apparently conformational epitopes by using linear peptide analogs have been in vain. In this study, conjugates of head-to-tail cyclic peptides encompassing the predicted top of a protective surface loop were used for immunization. A series of 18 cyclic peptides with a ring size ranging from 7 to 17 residues, conjugated to tetanus toxoid, was investigated. Antipeptide and anti-whole-cell immunoglobulin G (IgG) titers elicited by the conjugates were determined. Conjugates of three peptides, containing 14, 15, and 17 amino acid residues (peptides 7, 12, and 13, respectively), induced an anti-whole-cell titer when Quillaja saponin A was used as the adjuvant. When alum was used as the adjuvant, the conjugate of peptide 12 did not elicit an anti-whole-cell response. From the Quillaja saponin A group, some of the sera obtained with conjugates of peptides 7 and 12 and all sera obtained with the peptide 13 conjugate were bactericidal in vitro. None of the sera evoked with alum as the adjuvant showed bactericidal activity. Nonbactericidal sera contained IgG1 primarily, whereas bactericidal sera showed significant titers of IgG2a and IgG2b. Class 1 protein-derived synthetic cyclic peptides which are capable of eliciting bactericidal antibodies, such as peptide 13 derived from meningococcal strain H44/76, represent potential candidates for a (semi)synthetic vaccine against meningococcal disease.