1. Isolation of N-linked glycopeptides from plasma Yong Zhou 1, Ruedi Aebersold 2, and Hui Zhang 1,3 * 1 Institute for Systems Biology, Seattle, Washington 98103, USA 2 Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich and Faculty of Natural Sciences, University of Zurich, CH-8093, Switzerland 3 Department of Pathology, Johns Hopkins Medical Institutes, Baltimore, Maryland 21287, USA Yong Zhou 1, Ruedi Aebersold 2, and Hui Zhang 1,3 * 1 Institute for Systems Biology, Seattle, Washington 98103, USA 2 Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich and Faculty of Natural Sciences, University of Zurich, CH-8093, Switzerland 3 Department of Pathology, Johns Hopkins Medical Institutes, Baltimore, Maryland 21287, USA OVERVIEW OVERVIEW Here we’ve described the optimization of a recently developed Solid-Phase Extraction of (N-linked) Glycopeptides (SPEG) procedure for blood plasma analysis by spiking with [ 14 C]-labeled human glycoproteins, which significantly enhances the specificity and yield of the isolated N-linked glycopeptides. After the optimization of each step of the SPEG procedure as discussed above, one new flow of N-linked glycopeptide capture can be drawn out as in Figure 4. The experiments show that 25 µl of hydrazide resin, 10mM NaIO 4 for 1 hour at room temperature, and 1.5 µl PNGase F for 14 hours at 37 ˚C yield optimal recovery of N- linked glycopeptides from tryptic peptides coming from 20 µl of mouse blood plasma. The buffer system for trypsin digestion is a critical factor for SPEG specificity and efficiency, with 50% TFE giving the best results. Finally, pre-digestion of plasma proteins using trypsin was shown to be one effective way to significantly increase the yield of SPEG. These results will improve the sensitivity of discovery of clinical biomarkers from blood. [1] Hui Zhang, Xiao-jun Li, Daniel Martin and Ruedi Aebersold, Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nature Biotechnology. 2003, 21:660-666. METHODS METHODS RESULTS RESULTS REFERENCESREFERENCES 14 C-IODOACTAMIDE LABELING HUMAN GLYCOPROTEINS  -1-acid glycoprotein 1 (AG)(Positive Control: 2 Cys in; 2 Cys out) MALSWVLTVLSLLPLLEAQIPLCANLVPVPITNATLDQITGKWFYIASAFRNEEYNKSVQ EIQATFFYFTPNKTEDTIFLREYQTRQDQCIYNTTYLNVQRENGTISRYVGGQEHFAHLL ILRDTKTYMLAFDVNDEKNWGLSVYADKPETTKEQLGEFYEALDCLRIPKSDVVYTDWKK DKCEPLEKQHEKERKQEEGES  -1-antitrypsin (AT) (Negative Control: 0 Cys in; 1 Cys out) MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS LYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGF QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ INDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTV KVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA VLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK CONCLUSIONCONCLUSION 1. Monitoring each step of SPEG procedure using mouse plasma spiked with [ 14 C]-labeled human blood glycoprotein [ 14 C]-Radioactivity assay offers very similar pattern of coupling efficiency comparing with N-linked glycopeptides quantified with LC- MS/MS and isotopic labeling. Here, the coupling efficiency for different NaIO 4 oxidation conditions were determined by either direct [ 14 C]-radioactivity assay of uncoupled fractions post coupling (A) or captured N-linked glycopeptides quantified by LC-MS/MS and isotopic-labeling (B). (C), The CID spectrum of one peptide from spiked human AG with specific M/Z value (2021.8498) and sequence (QDQCIYN#TTIYNVQR). (D), The ASAPRatio of the peptide identified in (C)—it’s abundance in Light (1mM NaIO 4 ) is only 0.209  0.013 fold of Heavy (10mM NaIO 4 ), i.e. one fifth. 1) Trypsin Digestion: 2) Optimization of PNGase F release and peptide-level SPEG flow by [ 14 C] radioactivity monitoring A), Yield comparison: PNGase F released radioactivity from spiked [ 14 C]-AG between protein-level and peptide-level SPEG; B), Saturation curve of hydrazide resin for glycocapture of digested glycopeptides from 20  l plasma spiked with [ 14 C]-AG. Data shown as percentage of original radioactivity retained on hydrazide resin after overnight coupling; C), Yield of N-linked glycopeptides via peptide-level SPEG by different volume of Affi-gel hydrazide slurry; D), Yield of N-linked glycopeptides via peptide-level SPEG by different amount of PNGase F-- 1.5  l PNGase F (1,250 U, New England Biolabs unit) is enough for 20  l mouse plasma. A), Released [ 14 C] radioactive activities from beads by tryptic digestion; B), PNGase F released [ 14 C] radioactive activities; C), PNGase F unreleased [ 14 C] radioactive activities. INTROCUCTION INTROCUCTION There is growing interest in discovery of disease biomarkers from blood plasma. For this reason, quantitative analysis of plasma proteins has been the focus of different proteomic technologies. The challenges faced by all quantitative plasma proteomic methods include complexity and the high dynamic range of the plasma sample. Therefore, a desirable proteomic technique for plasma profiling must be sensitive, reproducible, and robust. Recently, we have developed a method for Solid-Phase Extraction of N-linked Glycopeptides (SPEG), and we have shown that analysis of plasma using SPEG improves dynamic range and sensitivity. Here, we optimized each step of the method and developed a standard procedure for plasma analysis using SPEG and mass spectrometry by spiking in two [ 14 C]-labeled human glycoproteins. Optimizing the recently developed Solid-Phase Extraction of (N-linked) Glycopeptides (SPEG) procedure for blood plasma analysis by spiking in [ 14 C]-labeled human glycoproteins significantly enhanced the specificity and yield of the isolated N-linked glycopeptides. This will improve the sensitivity of discovery of clinical biomarkers from blood. 3. Optimizing each step Here, five conditions were tested, including: 100 mM NH 4 HCO 3 ; 8M Urea in 100 mM NH 4 HCO 3 (original); 8M Urea in 100 mM NH 4 HCO 3, plus 2 mM CaCl 2 ; 0.1% RapiGest in 100 mM NH 4 HCO 3 ; and 50% TFE (Triflouroethonal) in 100 mM NH 4 HCO 3. 4. A flow chart illustrating the optimized peptide- level SPEG procedure 2. Evaluating the performance of the original SPEG procedure using mouse plasma spiked with [ 14 C]- labeled two human blood glycoproteins—AG and AT Hydrazide beads Glycans Oxidized glycans