Mass spectrometry based proteomics for absolute quantification of proteins from tumor cells

Abstract

In-depth quantitative profiling of the proteome and sub-proteomes of tumor cells has relevance to tumor classification, the development of novel therapeutics, and of prognostic and predictive markers and to disease monitoring. In particular the tumor cell surface represents a highly relevant compartment for the development of targeted therapeutics and immunotherapy. We have developed a proteomic platform to profile tumor cells that encompasses enrichment of surface membrane proteins, intact protein fractionation and label-free mass spectrometry based absolute quantification. Here we describe the methodology for capture, identification and quantification of cell surface proteins using biotinylation for labeling of the cell surface, avidin for capture of biotinylated proteins and ion mobility mass spectrometry for protein identification and quantification.

Keywords: Absolute quantification; Mass spectrometry; Proteins; Tumor cells.

Fig. 1

Quantitative MS based protein biomarker discovery platform employing the integration of in vivo cell-surface protein labeling, intact protein RP-HPLC fractionation and LC-HDMS^E for quantifying proteins from patient with AML.

Fig. 2

Reversed-phase HPLC fractionation of proteins prepared from leukemia cells. After trypsin digestion, 24 pooled fractions were analyzed by LC-HDMS^E. 380 μg of protein from TCE and 76 μg of protein from cell-surface were separated by a high performance liquid chromatography system (Shimadzu) controlled by EZ Start workstation (version 7.4 SP3) and equipped with UV detection at 280 nm. Proteins were separated on a RPGS reversed-phase column (4.6 mm I.D. × 150 mm, 15 μm, 1000 Å, Column Technology Inc.). The separation was performed via gradient elution of two mobile phases: 0.1% TFA in 95% H₂O and 0.1% TFA in 95% ACN at a constant flow rate of 2.1 mL/min. Fractions were collected at 20 s intervals. Pool-1: fraction # 1–22; Pool-2: fraction # 23–24; Pool-3: fraction # 25–26; Pool-4: fraction # 27–28; Pool-5: fraction # 29–30; Pool-6: fraction # 31–32; Pool-7: fraction # 33– 34; Pool-8: fraction # 35–36; Pool-9: fraction # 37–38; Pool-10: fraction # 39–40; Pool-11: fraction # 41–42; Pool-12: fraction # 43–44; Pool-13: fraction # 45–46; Pool-14: fraction # 47–48; Pool-15: fraction # 49–50; Pool-16: fraction # 51–52; Pool-17: fraction # 53–54; Pool-18: fraction # 55–56; Pool-19: fraction # 57–58; Pool-20: fraction # 59–60; Pool-21: fraction # 61–62; Pool-22: fraction # 63–64; Pool-23: fraction # 65–72; Pool-24: fraction # 73–84.

Fig. 3

TIC Chromatograms of RPLC-HDMS^E. (A) RPLC gradient elution; (B) low energy TIC chromatogram; (C) elevated energy TIC chromatogram. Approximately 1 μg of protein digest from each of 24 pooled fractions was loaded onto the trap-column (C18, 180 μm × 20 mm, 5 μm) through a 20 μL sample-loop using 98% mobile phase A (0.1% formic acid in 2% ACN) at a flow rate of 8 μL/min for 5 min. The desalted peptides eluted from the analytical column (C18, 75 μm × 150 mm, 1.8 μm) at a flow rate of 500 nL/min with a 120-min of gradient elution: from 3% to 25% mobile phase B (0.1% formic acid in 98% ACN) over 100 min, 25–85% mobile phase B for 7 min, a wash step to hold at 85% mobile phase B for 3 min, and a re-equilibration step at 3% mobile phase B for 10 min.

Fig. 4

Mass spectra of HDMS^E. (A): Low energy mass spectrum (peptide ions, HDMS); (B): high energy mass spectra (fragment ions, HDMS^E). LC-HDMS^E data was acquired in resolution mode with SYNAPT G2-S. The capillary voltage was set to 2.80 kV, sampling cone voltage to 30 V, source offset to 30 V, and source temperature to 100 °C. Mobility utilized high-purity N₂ as the drift gas in the IMS TriWave cell. IMS wave velocity was 600 m/s, helium cell DC was 50 V, Trap DC bias was 45 V, IMS TriWave DC bias was 3 V, and IMS wave delay was 1000 μs. The mass spectrometer was operated in V-mode with a resolving power of at least 20,000. Argon was used as the collision gas. In low energy HDMS mode, data was collected at collision energy of 2 eV in both Trap cell and Transfer cell. In high energy HDMS^E mode, the collision energy was ramped from 25 to 55 eV in the Transfer cell.

Fig. 5

Absolute quantification of proteins from two different cellular compartments (cell-surface and total cell extract, TCE) of a leukemia cell population. (A) Patient #1_Cell surface; (B) Patient #2_Cell surface; (C) Patient #1_TCE; (D) Patient #2_TCE. Axis X is the number of quantified proteins, axis Y is the quantified protein amount (fmol on column) combined from the LC-HDMS^E analysis of 24 pooled fractions.

Fig. 6

Comparison of two types of plasma membrane proteins quantified from cell-surface sample and total cell extract (TCE) sample. (A): CD antigens; (B): HLA class I/II histocompatibility antigens.