Instrumentation

The PRF is equipped with two high-resolution mass spectrometers (ThermoFisher Scientific) devoted exclusively for proteomic analysis. While both instruments are capable of unequivocal identification and quantification of proteins and post-translational modifications from complex mixtures, their different architecture makes them uniquely suited for different proteomic approaches.

Orbitrap Fusion Tribrid with ETD

(Acquired with funding from the Protein Folding Disorder Initiative, a part of University of Michigan’s Strategic Research Initiative).

• The Thermo Scientific™ Orbitrap Fusion™ Tribrid™ high-resolution mass spectrometer combines the best of quadrupole, Orbitrap, and ion trap mass analysis in a revolutionary Tribrid architecture that delivers unprecedented depth of analysis. It enables life scientists analyzing even the most challenging low-abundance, high-complexity, or difficult samples to identify more compounds more quickly, quantify more accurately, and elucidate structures more thoroughly. The instrument can perform parallel CID, HCD, ETD, EThcD and ETciD fragmentation analysis to provide the most in-depth information about the complex samples. With its ability to perform multinotch-MS3 (SPS-MS3), Orbitrap Fusion provides more accurate results when using Tandem Mass Tag (TMT) approach for relative quantitation.

In its current setup, Orbitrap Fusion is used for standard qualitative and SPS-MS3 based relative quantitation using TMT.

Schematic of the Orbitrap Fusion Tribrid MS (taken from http://planetorbitrap.com).

Q Exactive HF

• The Thermo Scientific™ Q Exactive HF is a hybrid quadrupole-Orbitrap mass spectrometer featuring an ultra-high-field Orbitrap analyzer. While it is capable of only HCD fragmentation, QE HF is faster and provides better resolution for every scan. Apart from its use in performing standard data-dependent acquisition (DDA), QE HF can also be used for data-independent acquisition (DIA) and parallel reaction monitoring (PRM; targeted proteomics)

In its current setup QE HF is used for standard qualitative, DIA, PRM proteomic approaches.

Note: Both mass spectrometers are equipped with dedicated RSLC-nano (Dionex) UHPLCs and nanospray sources.

1260 Infinity II ANALYTICAL HPLC (Agilent)

• This analytical HPLC is used exclusively for off-line fractionation of the complex peptide mixture. Each of these fractions is further separated by a column in-line with the mass spectrometer providing a robust two-dimensional (2D) separation of peptides. This often provides more in-depth coverage of the proteome.