Off-Line Two-Dimensional Liquid Chromatography with Maximized Sample Loading to Reversed-Phase Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry for Shotgun Proteome Analysis

Abstract

We demonstrate a strategy of maximizing the performance of reversed-phase (RP) liquid chromatography (LC) tandem mass spectrometry (MS/MS) for efficient shotgun proteome analysis by optimizing the sample loading to the instrument in an off-line two-dimensional (2D) LC tandem MS platform. To determine the quantity of peptides present in a proteome digest or fractionated peptides from strong-cation exchange (SCX) separation, an automated system based on RPLC with a rapid step solvent gradient for peptide elution and ultraviolet (UV) detection was developed. This system also allowed the purification of the peptides by removing salts and other impurities present in a sample. It was found that controlling the amount of peptides injected into a RPLC MS/MS system was critical to achieve the maximum efficiency in peptide and protein identification. With the use of off-line 2D-LC-MS/MS, peptide fractions from the first dimension of separation were desalted and quantified, followed by injecting the optimal amount of the sample into RPLC-MS/MS for peptide sequencing. The application of this strategy was demonstrated in the proteome profiling of breast cancer MCF-7 cells. From the analysis of 28 SCX fractions with each injecting 1 microg of sample into a 75 mum x 100 mm C18 column interfaced to a quadrupole/time-of-flight mass spectrometer, a total of 2362 unique proteins or protein groups were identified with a false positive peptide identification rate of 0.19%, as determined by target-decoy proteome sequence searches. Replicate 2 h runs of individual fractions with the exclusion of precursor ions of peptides already identified in the first runs resulted in the identification of an additional 549 unique proteins or protein groups with a false positive identification rate of 0.60%. This example illustrated that off-line 2D-LC-MS/MS, with maximal sample injection to the RPLC-MS, is an effective method for shotgun proteome analysis. Finally, the advantages and limitations of this method, compared to other methods, are discussed.

Proteome-Wide Identification of Novel Binding Partners to the Oncogenic Fusion Gene Protein, NPM-ALK, using Tandem Affinity Purification and Mass Spectrometry

“…7 In addition to the use of TAP, we also used our recently developed MS method to enhance the detection and identification of the interacting proteins. This highly sensitive technique is based on the use of precursor ion exclusion 24 and optimized sample loading 25 in a capillary liquid chromatography (LC) quadrupole time-of-flight mass spectrometer. A replicate LC-MS run of the same sample with precursor ion exclusion allows identification of low abundance peptides by excluding the high abundance peptides identified in the first run.…”

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

“…Thus, to gain a general overview of the mouse adipose tissue proteome, we investigated integral mouse WAT samples without preceding separation of the distinct tissue fractions. Salt-based gradient SCX chromatography displays excellent orthogonality with reverse phase peptide separation [49] and has successfully been employed in the preparation of many complex proteomic samples over the past several years [27,[50][51][52]. However, the exploitation of this classical, salt-based gradient SCX elution is significantly limited due to (i) low robustness of the system, (ii) ionization suppression effects, and (iii) peptides overlapping between the fractions.…”

“…However, the exploitation of this classical, salt-based gradient SCX elution is significantly limited due to (i) low robustness of the system, (ii) ionization suppression effects, and (iii) peptides overlapping between the fractions. To avoid the frequent autosampler blockage and capillary clogging in nanoscale RP chromatography caused by the high salt concentration, a buffer cleanup step is required [51] system robustness. To overcome these weak points, we employed a pH-based SCX gradient approach and compared its performance for adipose tissue proteome analysis.…”

Studies of Phosphoproteomic Changes Induced by Nucleophosmin-Anaplastic Lymphoma Kinase (ALK) Highlight Deregulation of Tumor Necrosis Factor (TNF)/Fas/TNF-related Apoptosis-induced Ligand Signaling Pathway in ALK-positive Anaplastic Large Cell Lymphoma

“…The beads were washed twice with 100 l of immunoaffinity purification buffer and twice with rinse buffer (100 mM Tris and 100 mM NaCl). The phosphotyrosine-containing peptides were eluted from the beads by using 0.1% TFA, desalted, quantified by UV absorbance (45), and then subjected to reverse phase LC/MS/MS analysis.…”