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Capillary electrophoresis coupled to electrospray ionization tandem mass spectrometry (CE-MS/MS) for ultra-sensitive proteomic analysis of limited sample

ABSTRACT: In this work, we developed an ultra-sensitive CE-MS/MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 pg and 88 pg of HeLa protein digest standard by CE-MS/MS yielded ~1,100±46 and ~160±59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-of-the-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultra-sensitive CE-MS/MS method for analysis of limited biological samples, we digested 500 and 1,000 HeLa cells using a miniaturized in-solution digestion workflow. From 1-, 5-, and 10-cell equivalents injected from the resulted digests, we identified 744±127, 1,139±24, and 1,271±6 proteins and 3,353±719, 5,709±513, and 8,527±114 peptide groups, respectively. Furthermore, we performed a comparative assessment of CE-MS/MS and two reversed-phased nano-liquid chromatography (RP-nLC-MS/MS) methods (monolithic and packed columns) for the analysis of a ~10 ng HeLa protein digest standard. Our results demonstrate complementarity in the protein-, and especially peptide-level identifications of the evaluated CE-MS- and RP-nLC-MS-based methods. The techniques were further assessed to detect post-translational modifications and highlight the strengths of the CE-MS/MS approach in identifying potentially important and biologically relevant modified peptides. With a migration window of ~60 min, CE-MS/MS identified ~2,000±53 proteins on average from a single injection of ~8.8 ng HeLa protein digest standard. Additionally, an average of 232±10 phosphopeptides and 377±14 N-terminal acetylated peptides were identified in CE-MS/MS analyses at this sample amount corresponding to 2- and 1.5-fold more identifications for each respective modification found by nLC-MS/MS methods.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Kendall Johnson

LAB HEAD: Alexander R. Ivanov

PROVIDER: PXD027226 | Pride | 2022-02-17

REPOSITORIES: Pride

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Capillary Electrophoresis Coupled to Electrospray Ionization Tandem Mass Spectrometry for Ultra-Sensitive Proteomic Analysis of Limited Samples.

Johnson Kendall R KR Greguš Michal M Kostas James C JC Ivanov Alexander R AR

Analytical chemistry 20220104 2

In this work, we developed an ultra-sensitive CE-MS/MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 and 88 pg of the HeLa protein digest standard by CE-MS/MS yielded ∼1100 ± 46 and ∼160 ± 59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-of-the-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultra ...[more]

PMID: 34983182

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Project description:In the light of the ongoing single-cell revolution, scientific disciplines are combining forces with the ultimate goal to retrieve as much relevant data as possible from trace amounts of biological material. For single cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, LC separation, MS/MS data acquisition and data analysis. To demonstrate the potential for single cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar array columns with state-of-the-art Orbitrap MS/MS detection and FAIMS. As compared to the currently commercially available pillar array columns, this dedicated limited sample column has a reduced cross section (factor of 2) and micro pillar dimensions that have been further downscaled (also by a factor of 2; 2.5 µm instead of 5 µm diameter), resulting in improved chromatography at reduced void times. A dilution series ranging from 5 to 0.05 ng/µL was prepared using trace amounts of PEG in the sample solvent to reduce adsorptive effects in the autosampler vial, sample stability up to 24h after dilution was demonstrated. Comparative processing of the MS/MS data with different database search algorithms (with and without second search feature activated and with and without rescoring based on fragmentation pattern prediction) pointed out the benefits of using Sequest HT together with INFERYS when analyzing samples at a concentration below 1 ng/µL. On average (data from quadruplicate runs), we were able to successfully identify 2855 unique protein groups from just 1 ng of HeLa lysate and this using a 60 min non-linear LC solvent gradient at a flow rate of 250 nL/min, hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10 (2436 proteins identified from 10 ng of HeLa lysate in 2019). By successfully identifying 1486 and 2210 proteins (average values, quadruplicates) from as little as 250 and 500 pg of HeLa lysate respectively, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.

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