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 reversephase 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.

MS-based proteomics has emerged as a powerful tool in biological studies. The shotgun proteomics strategy, in which proteolytic peptides are analyzed in data-dependent mode, enables a detection of the most comprehensive proteome (>10 000 proteins from whole-cell lysate). The quantitative proteomics uses stable isotopes or label-free method to measure relative protein abundance. The isotope labeling strategies are more precise and accurate compared to label-free methods, but labeling procedures are complicated and expensive, and the sample number and types are also limited. Sequential window acquisition of all theoretical mass spectra (SWATH) is a recently developed technique, in which data-independent acquisition is coupled with peptide spectral library match. In principle SWATH method is able to do label-free quantification in an MRM-like manner, which has higher quantification accuracy and precision. Previous data have demonstrated that SWATH can be used to quantify less complex systems, such as spiked-in peptide mixture or protein complex. Our study first time assessed the quantification performance of SWATH method on proteome scale using a complex mouse-cell lysate sample. In total 3600 proteins got identified and quantified without sample prefractionation. The SWATH method shows outstanding quantification precision, whereas the quantification accuracy becomes less perfect when protein abundances differ greatly. However, this inaccuracy does not prevent discovering biological correlates, because the measured signal intensities had linear relationship to the sample loading amounts; thus the SWATH method can predict precisely the significance of a protein. Our results prove that SWATH can provide precise label-free quantification on proteome scale.

In addition to O-phosphorylation, O-linked modifications of serine and threonine by ␤-N-acetyl-D-glucosamine (GlcNAc) may regulate muscle contractile function. This study assessed the potential role of O-GlcNAcylation in cardiac muscle contractile activation. To identify specific sites of O-GlcNAcylation in cardiac myofilament proteins, a recently developed methodology based on GalNAz-biotin labeling followed by dithiothreitol replacement and light chromatography/tandem mass spectrometry site mapping was adopted. Thirty-two O-GlcNAcylated peptides from cardiac myofilaments were identified on cardiac myosin heavy chain, actin, myosin light chains, and troponin I. To assess the potential physiological role of the GlcNAc, force- [Ca 2؉ ] relationships were studied in skinned rat trabeculae. Exposure to GlcNAc significantly decreased calcium sensitivity (pCa50), whereas maximal force (F max ) and Hill coefficient (n) were not modified. Using a pan-specific O-GlcNAc antibody, it was determined that acute exposure of myofilaments to GlcNAc induced a significant increase in actin O-GlcNAcylation. This study provides the first identification of O-GlcNAcylation sites in cardiac myofilament proteins and demonstrates their potential role in regulating myocardial contractile function. D iabetes mellitus is a risk factor for the development of heart failure, 1 and abnormal glucose metabolism may contribute directly to depressed cardiac function. Studies in humans and animal models of diabetes mellitus have demonstrated abnormal myofilament function 2 and impaired excitation-contraction coupling, 3,4 which may depress myocardial function. Posttranslational modifications of myofilament proteins regulate cardiac function and phosphorylation of myofilament proteins may result in functional abnormalities in heart failure. [5][6][7] In addition to O-linked phosphorylation of serine (Ser) and threonine (Thr) residues of proteins, dynamic Materials and Methods Mass Spectrometric Identification of O-GlcNAc-Modified ProteinsTo label the specific sites (further details are in the online data supplement, available at http://circres.ahajournals.org), GlcNAcmodified peptides were labeled with GalNAz-biotin and enriched by avidin chromatography, and then dithiothreitol (DTT) was used to replace the GlcNAc-GalNAz-biotin by ␤-elimination and Michael addition (BEMAD) as previously described. 14 Isolated Skinned Fiber StudiesFor skinned fiber studies, cardiac trabeculae were isolated and mounted as previously described. 5 ImmunoblotsMyofilament proteins were isolated as previously described, 15 with minor modifications. To determine the global GlcNAc modifications of myofilament proteins, a pan-GlcNAc antibody (CTD 110.6, Covance) was used as previously described. 16 To assess cardiac troponin I (cTnI) phosphorylation, a phospho-TnI (Ser23/Ser24) antibody (Cell Signaling, Danvers, Mass) was used as previously described. 5 Results and Discussion Myofilament Proteins Are Modified by O-GlcNAcWith the enrichment and BEMAD experiments describ...