Peptide labeling with isobaric tags has become a popular technique in quantitative shotgun proteomics. Using two different samples viz. a protein mixture and HeLa extracts, we show that three commercially available isobaric tags differ with regard to peptide identification rates: The number of identified proteins and peptides was largest with iTRAQ 4-plex, followed by TMT 6-plex, and smallest with iTRAQ 8-plex. In all experiments, we employed a previously described method where two scans were acquired for each precursor on an LTQ Orbitrap: A CID scan under standard settings for identification, and a HCD scan for quantification. The observed differences in identification rates were similar when data was searched with either Mascot or Sequest. We consider these findings to be the result of a combination of several factors, most notably prominent ions in CID spectra as a consequence of loss of fragments of the label tag from precursor ions. These fragment ions cannot be explained by current search engines and were observed to have a negative impact on peptide scores.

We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO 2 )-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a twodimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO 2 -filled tips. To increase the trapping selectivity of the TiO 2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient. INTRODUCTIONThe majority of biochemical processes are induced and controlled by post-translational modifications on certain proteins. One of the most frequent modifications is phosphorylation of the amino acids-serine, threonine and tyrosine. It is this reversible phosphorylation that regulates the major (or even the majority of) cellular processes, and it has been estimated that almost 30% of all proteins in mammalian cells are phosphorylated at some point during their expression 1,2 . This alone would be reason enough to invest time and resources in the analysis of phosphopeptides.Despite the fact that there are so many phosphoproteins in the cell, phosphorylated residues remain at very low concentrations physiologically and are present in substoichiometric quantities. The presence of high-abundance peptides in samples of biological origin makes it necessary to develop an efficient separation and enrichment method for phosphopeptides.Earlier established methods for phosphorylation site detection relied on Edman degradation of 32 P-labeled peptides. Edman degradation is a robust and well-established method, but it shows some limitations when analyzing complex samples and cannot fulfill the requirements for sensitive detection of low concentrations of phosphopeptides [1][2][3] . Besides the additional needed labeling of the phosphate residues, it would take more than a hundred fold in time to chemically treat and analyze a complex sample related to other techniques for sequencing and phosphorylation site mapping.Identifying phosphorylation sites in different proteins through mass spectrometry (MS) requires a proper pre-separation and enrichment of the phosphopeptides performed online by the HPLC, because of the often highly complex sample composition.Some phosphopeptide-selective techniques have been developed, such as immobilized metal affinity chromatography (IMAC) [4][5][6] or two-dimensional HPLC systems using strong cation...