Protein Chemistry and Proteomics Shared Resource

The Protein Chemistry and Proteomics Shared Resource supports, in part, personnel, services, and operations of the Mayo Proteomics Research Center (MPRC), and is a vital component of the Mayo Clinic Cancer Center. Its mission is to provide the skills, tools, and methods to foster a complete understanding of the human proteome and the translation of this knowledge to predict, prevent, and cure human disease (e.g. cancer).

In the five year period following its formation from the merge of the Mayo Protein Core Facility with the Biomedical Mass Spectrometry Facility in 2002, the MPRC initiated a rapid expansion of core services and the development of new methodologies and instrumentation (e.g. FT-ICR MS and 2-D difference in-gel electrophoresis or DIGE). The MPRC supports a broad range of programs and projects in cancer research, including investigations in biomarker discovery, differential proteomics, post-translation modification determinations, examination of non-covalent interactions, protein identification and small molecule analysis.

Currently, MPRC provides Mayo Clinic Cancer Center members the following services:

• Solid phase peptide synthesis by Fmoc orthogonal protection chemistry
• Synthesis of peptide/protein specific immunogens on KLH or BSA carrier proteins
• Edman N-terminal chemical protein sequencing
• Quantitative amino acid analysis of proteins and peptides
• Protein separation and purification by reverse phase HPLC and FPLC methods
• Computer-aided analysis of protein sequences for design of synthetic peptides and custom anti-peptide antibodies
• De Novo design of protein models utilizing Tripos' Sybyl 7.3 and Advanced Protein Modeling software
• 1-Dimensional and 2-Dimensional PAGE of complex protein mixtures including 2-D DIGE separation
• Protein identification by nanoLC-MS/MS methods
• Differential Proteomics / Biomarker discovery by tandem MS and targeted MS (e.g. SRM, SIM) methods
• Small molecule analysis by LC-MS
• Protein PTM analysis by nanoLC-FT-ICR mass spectrometry

Facilities, Equipment, and Staffing

The MPRC is well-equipped, housing three LTQ-Orbitrap mass spectrometers, an LTQ FT-ICR (Fourier Transform Ion Cyclotron Resonance) instrument, a Q-TOF (Quadrupole Time-of-Flight) and two TSQ Quantum triple quadrupole mass spectrometers, two ESI-TOF instruments, and an ABI 4800 TOF/TOF mass spectrometer that is shared with the University of Minnesota. In addition, the Peptide Synthesis Laboratory of the MPRC houses several major pieces of instrumentation including three ABI 433A Peptide Synthesizers with SynthAssist monitoring software, two AAPPTEC APEX 396 Multiple Peptide Synthesizers, two CEM Liberty Microwave Peptide Synthesizers, a MSQ mass spectrometer, two Hewlett Packard Analytical RP-HPLC systems and two Waters Preparative RP-HPLC systems.

Significant computing/data management resources are available, including over 20 Terabytes of data storage space available and a 60 processor Linux cluster utilized for data analysis and database searching. Rosetta Elucidator software has been implemented for label-free differential analysis in addition to our own in-house developed RAAMS software (Mol Cell. Proteomics 6:305-318, 2007 for analysis of 18O labeled peptides. Software available for database searching includes Mascot, Sequest, X! Tandem, Scaffold, and PEAKS de novo protein identification tools. In addition, four full-time programmers provide software and data analysis support to develop new software and algorithms for proteomics and biomarker analysis of samples from cancer-related investigations and patients with evidence of neoplastic disease.

In addition to the above, the MPRC shared resource also provides basic services in protein chemistry, which include:

• Solid phase peptide synthesis
• Protein purification and quantification
• Amino acid analysis
• Chemical protein sequencing
• Protein separation by 2-D polyacrylamide gel electrophoresis (PAGE)
• Protein/peptide molecular modeling