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Probing the complementarity of FAIMS and strong cation exchange chromatography in shotgun proteomics.

ABSTRACT: High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) offers benefits for the analysis of complex proteomics samples. Advantages include increased dynamic range, increased signal-to-noise, and reduced interference from ions of similar m/z. FAIMS also separates isomers and positional variants. An alternative, and more established, method of reducing sample complexity is prefractionation by use of strong cation exchange chromatography. Here, we have compared SCX-LC-MS/MS with LC-FAIMS-MS/MS for the identification of peptides and proteins from whole cell lysates from the breast carcinoma SUM52 cell line. Two FAIMS approaches are considered: (1) multiple compensation voltages within a single LC-MS/MS analysis (internal stepping) and (2) repeat LC-MS/MS analyses at different and fixed compensation voltages (external stepping). We also consider the consequence of the fragmentation method (electron transfer dissociation or collision-induced dissociation) on the workflow performance. The external stepping approach resulted in a greater number of protein and peptide identifications than the internal stepping approach for both ETD and CID MS/MS, suggesting that this should be the method of choice for FAIMS proteomics experiments. The overlap in protein identifications from the SCX method and the external FAIMS method was ~25% for both ETD and CID, and for peptides was less than 20%. The lack of overlap between FAIMS and SCX highlights the complementarity of the two techniques. Charge state analysis of the peptide assignments showed that the FAIMS approach identified a much greater proportion of triply-charged ions.

SUBMITTER: Creese AJ

PROVIDER: S-EPMC3586169 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This dataset contains the raw data from the JASMS paper with the same title and DOI: 10.1007/s13361-012-0544-2. We compared the results of shotgun proteomic experiments of a trypsin digest of SUM52 cell lysate using 2D-LC fractionated (strong cation exchange and reversed phase of 6 SCX fractions) with FAIMS based fractionation. The FAIMS fractionation was performed using two different methods, an internal stepping method where the compensation voltage (CV) is cycled through 6 different voltages during the LC run (the three most abundant ions are fragmented at each CV. To match with the other methods this was run in six replicates. The other method was termed external stepping, for this method individual LC runs were performed at the six CV’s used earlier (-25 V to -50 V in 5 V steps). The three most abundant ions were again fragmented. For all three of these methods data was collect using electron transfer dissociation (ETD) and collision induced dissociation (CID) giving a total of 6 datasets with 6 raw files in each. SUM52 breast cancer carcinoma cells were cultured in HPMi-1640 formulation, supplemented with 2 mM L-glutamine, 1% Pen-Strep and 10% PBS at 37 oC in a 5% CO2 atmosphere. Lysis buffer contained Triton X-100 (0.5%), NaCl (0.15 M), PhosphoStop phosphatase inhibitor tablet (Roche) and Mini-complete protease inhibitor (Roche). The lysed cells were digested with trypsin at a ratio of 50:1 protein to enzyme. Part of the digest was fractionated with strong cation exchange using a Polysulfoethyl A column. All fractions and crude digest were desalted prior to mass spectrometry analysis. Approximately 1.66 µg of digest was loaded per LC-MS/MS run onto a 150mm Acclaim PepMap100 C18 column and separated over a 30 minute gradient from 3.2% acetonitrile (0.1% formic acid) to 44% acetonitrile (0.1% formic acid). The raw files were searched using proteome discoverer 1.2 against the human IPI database V3.81 supplemented with known contaminants and the reverse sequences. The files were searched with both sequest and mascot (v2.2.0). The MSF files were manually filtered using mascot significance scores and XCORR vs change state to give a 1% FDR, for filter setting see the linked paper. Included in the downloadable files is a xls file which documents the raw files listed and the names they had for the original searches, the names have been changed for clarity of the dataset.

Project description:Two-dimensional (2D) liquid chromatography (LC)-tandem mass spectrometry (MS/MS) are typically employed for deep bottom-up proteomics, and the state-of-the-art 2D-LC-MS/MS has approached over 8000 protein identifications (IDs) from mammalian cell lines or tissues in 1-3 days of mass spectrometer time. Capillary zone electrophoresis (CZE)-MS/MS has been suggested as an alternative to LC-MS/MS for bottom-up proteomics. CZE-MS/MS and LC-MS/MS are complementary in protein/peptide ID from complex proteome digests because CZE and LC are orthogonal for peptide separation. In addition, the migration time of peptides from CZE-MS can be predicted accurately, which is invaluable for evaluating the confidence of peptide ID from the database search and even guiding the database search. However, the number of protein IDs from complex proteomes using CZE-MS/MS is still much lower than the state of the art using 2D-LC-MS/MS. In this work, for the first time, we established a strong cation exchange (SCX)-reversed phase LC (RPLC)-CZE-MS/MS platform for deep bottom-up proteomics. The platform identified around 8200 protein groups and 65,000 unique peptides from a mouse brain proteome digest in 70 h. The data represents the largest bottom-up proteomics dataset using CZE-MS/MS and provides a valuable resource for further improving the tool for prediction of peptide migration time in CZE. The peak capacity of the orthogonal SCX-RPLC-CZE platform was estimated to be around 7000. SCX-RPLC-CZE-MS/MS produced comparable numbers of protein and peptide IDs with 2D-LC-MS/MS (8200 vs. 8900 protein groups, 65,000 vs. 70,000 unique peptides) from the mouse brain proteome digest using comparable instrument time. This is the first time that CZE-MS/MS showed its capability to approach comparable performance to the state-of-the-art 2D-LC-MS/MS for deep proteomic sequencing. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein and peptide IDs and combining those two methods improved the number of protein group and unique peptide IDs by nearly 10% and over 40%, respectively, compared with 2D-LC-MS/MS alone.

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Probing the complementarity of FAIMS and strong cation exchange chromatography in shotgun proteomics.

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