title: High pH Reversed-Phase Micro-Columns for Simple, Sensitive, and Efficient Fractionation of Proteome and (TMT labeled) Phosphoproteome Digests.
abstract: Despite recent advances in mass spectrometric sequencing speed and improved sensitivity, the in-depth analysis of proteomes still widely relies on off-line peptide separation and fractionation to deal with the enormous molecular complexity of shotgun digested proteomes. While a multitude of methods has been established for off-line peptide separation using HPLC columns, their use can be limited particularly when sample quantities are scarce. In this protocol, we describe an approach which combines high pH reversed-phase peptide separation into few fractions in StageTip micro-columns. This miniaturized sample preparation method enhances peptide recovery and hence improves sensitivity. This is particularly useful when working with limited sample amounts obtained from e.g., phosphopeptide enrichments or tissue biopsies. Essentially the same approach can also be applied for multiplexed analysis using tandem mass tags (TMT) and can be parallelized in order to deliver the required throughput. Here, we provide a step-by-step protocol for TMT6plex labeling of peptides, the construction of StageTips, sample fractionation and pooling schemes adjusted to different types of analytes, mass spectrometric sample measurement, and downstream data processing using MaxQuant. To illustrate the expected results using this protocol, we provide results from an unlabeled and a TMT6plex labeled phosphopeptide sample leading to the identification of >17,000 phosphopeptides in 8 h (Q Exactive HF) and >23,000 TMT6plex labeled phosphopeptides (Q Exactive Plus) in 12 h of measurement time. Importantly, this protocol is equally applicable to the fractionation of full proteome digests.
authors.affiliation: Technische Universität München, Emil Erlenmeyer Forum 5, 85354, Freising, Germany.|Center for Integrated Protein Science Munich (CIPSM), Freising, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ruprecht
authors.affiliation: Technische Universität München, Emil Erlenmeyer Forum 5, 85354, Freising, Germany.|German Cancer Consortium (DKTK), Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Zecha
authors.affiliation: Technische Universität München, Emil Erlenmeyer Forum 5, 85354, Freising, Germany.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Zolg
authors.affiliation: Technische Universität München, Emil Erlenmeyer Forum 5, 85354, Freising, Germany. kuster@tum.de.|Center for Integrated Protein Science Munich (CIPSM), Freising, Germany. kuster@tum.de.|German Cancer Consortium (DKTK), Heidelberg, Germany. kuster@tum.de.|German Cancer Research Center (DKFZ), Heidelberg, Germany. kuster@tum.de.|Bavarian Bimolecular Mass Spectrometry Center, Technische Universität München, Freising, Germany. kuster@tum.de.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuster
mesh_terms: D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase / Q000379:methods; D030562:Databases, Protein; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D013223:Statistics as Topic; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome
keywords: Fractionation; Isotope labeling; Mass spectrometry; Phosphorylation; Proteomics; Sample preparation