Mass spectrometry in cancer biomarker research: a case for immunodepletion of abundant blood-derived proteins from clinical tissue specimens

Abstract

The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear cell renal cell carcinoma as a model disease. Integrative analysis of data from this approach and genomic data obtained from the same type of tumor revealed concordant key pathways and protein targets germane to clear cell renal cell carcinoma. This includes the activation of the lipogenic pathway characterized by increased expression of adipophilin (PLIN2) along with 'cadherin switching', a phenomenon indicative of transcriptional reprogramming linked to renal epithelial dedifferentiation. We also applied immunodepletion of abundant blood-derived proteins to various tissue types (e.g., adipose tissue and breast tissue) showing unambiguously that the removal of abundant blood-derived proteins represents a powerful tool for the reproducible profiling of tissue proteomes. Herein, we show that the removal of abundant blood-derived proteins from solid tissue specimens is of equal importance to depletion of body fluids and recommend its routine use in the context of biological discovery and/or cancer biomarker research. Finally, this perspective presents the background, rationale and strategy for using tissue-directed high-resolution/accuracy MS-based shotgun proteomics to detect genuine tumor proteins in the peripheral blood of a patient diagnosed with nonmetastatic cancer, employing concurrent liquid chromatography-MS analysis of immunodepleted clinical tissue and blood specimens.

Figure 1. Experimental design

LC: Liquid chromatography; LC-LIT-FTICR-MS: Liquid chromatography linear ion trap Fourier transform ion cyclotron mass spectrometer; SCX: Strong cation exchange.

Figure 2. Distribution of blood, lymph and interstitial fluid in human tissue

Tissue-embedded networks of blood and lymph capillaries along with interstitial fluid compartment contain significant amounts of abundant blood-derived proteins. Reproduced with permission from [104].

Figure 3. SDS-PAGE analysis of immunoaffinity-depleted renal cell carcinoma tissue homogenate and matched patient serum

(A) Renal cell carcinoma tissue homogenate; (B) matched patient serum. Samples were resolved on a 4–12% Bis-Tris gel (Invitrogen Life Technologies, CA, USA) and stained with a SilverQuest™ Silver Staining Kit (Invitrogen Life Technologies). (A) Lane designations: lane 1, normal kidney tissue homogenate (5 µg); lane 2, RCC tissue homogenate (5 µg); lane 3, depleted normal kidney tissue homogenate (5 µg); and lane 4, depleted RCC tissue homogenate (5 µg). (B) Lane designations: lane 1, renal cell carcinoma patient serum (5 µg); lane 2, depleted matched patient serum (5 µg); and lane 3: high-abundant fraction (15 µl).

Figure 4. Clinical tissue immunodepletion workflow

Each surgically collected fresh frozen tissue block was sectioned into 8-µm-thick slices using a cryostat followed by tip sonication and bicinchoninic acid assay. Particulates were removed from the homogenate using a 0.22-µm spin filter. A total of 1000 µg of tissue protein was immunodepleted using MARS Human 14 immunoaffinity cartridges (Agilent, CA, USA). The resulting low-abundant protein fractions were pooled and concentrated using 5 kDa molecular weight cutoff spin concentrators (Agilent, CA, USA). Immunodepleted tissue homogenate (200 µg each) was then digested with trypsin. Final clean-up of the tryptic digest was achieved using a C18 column (Waters, MA, USA). Peptide fractions were collected using off-line SCX chromatography and analyzed by µRPLC tandem mass spectrometry. QC measures were employed to assess MARS Human 14 column (Agilent) integrity by removing an aliquot of both the low- and high-abundant pool and subsequently resolving the proteins on an SDS-PAGE gel. µRPLC: Microflow reversed-phase liquid chromatography; LC: Liquid chromatography; MARS: Multiple Affinity Removal System; MS/MS: Tandem mass spectrometry; QC: Quality control; SCX: Strong cation exchange.

Figure 5. Detecting adipophilin in renal cell carcinoma patient-derived tissue lysates using western blotting

Paired tissue lysates from two patients (lane 1: renal cell carcinoma [RCC], patient 1; lane 2: normal renal tissue, patient 1; lane 3: RCC, patient 2; lane 4: normal renal tissue, patient 2) were used. Quantitative analysis of the blot as fold change is shown in the lower panel. The blot intensity from the RCC patient was normalized to the blot intensity from the respective normal renal tissue. Elevated adipophilin was detected in RCC compared with the respective normal renal tissues. Tissue lysates were purchased from Abcam (MA, USA). A total of 10 µg of tissue lysates were separated on 4–20% Tris-glycine gradient gels (Invitrogen Life Technologies, CA, USA), transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, CA, USA) and blotted with anti-adipophilin antibody (Epitomics, CA, USA) followed by a peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, PA, USA). Image of the blot was quantitated using Image J (NIH, MD, USA).

Figure 6. SDS-PAGE analysis of immunoaffinity-depleted Ewing's sarcoma tissue homogenate and matched patient serum

(A) Ewing's sarcoma (EWS) tissue homogenate; (B) matched patient serum. Samples were resolved on a 4–12% Bis-Tris gel (Invitrogen Life Technologies, CA, USA) and stained with SimplyBlue™ Coomassie^® G-250 SafeStain (Invitrogen Life Technologies). (A) Lane designations: lane 1, EWS tissue homogenate (10 µg); lane 2, depleted EWS tissue homogenate (10 µg); and lane 3, high-abundant fraction (20 µl). (B) Lane designations: lane 1, matched patient serum (10 µg); lane 2, depleted patient serum (20 µl); and lane 3, high-abundant fraction (20 µl).

Figure 7. SDS-PAGE analysis of immunoaffinity-depleted breast tissue homogenate and matched patient serum

(A) Breast tissue homogenate; (B) matched patient serum. Samples were resolved on a 4–12% Bis-Tris gel (Invitrogen Life Technologies, CA, USA) and stained with SimplyBlue™ Coomassie^® G-250 SafeStain (Invitrogen Life Technologies). (A) Lane designations: lane 1, SeeBlue^® Plus2 (Invitrogen Life Technologies) prestained protein molecular weight standard (kDa); lane 2, breast tissue homogenate (15 µg); lane 3, depleted breast tissue homogenate, flow-through fraction #1 (20 µl); lane 4, depleted breast tissue homogenate, flow-through fraction #2 (20 µl); and lane 5, high-abundant fraction (20 µl). (B) Lane designations: lane 1, SeeBlue Plus2 prestained protein molecular weight standard (kDa); lane 2, matched patient serum (10 µg); lane 3, depleted patient serum, flow-through fraction #1 (20 µl); lane 4, depleted patient serum, flow-through fraction #2 (20 µl); and lane 5, high-abundant fraction (20 µl).

Figure 8. SDS-PAGE analysis of immunoaffinity-depleted adipose tissue homogenate and matched patient serum

(A) Adipose tissue homogenate; (B) matched patient serum. Samples were resolved on a 4–12% Bis-Tris gel (Invitrogen Life Technologies, CA, USA) and stained with SimplyBlue™ Coomassie^® G-250 SafeStain (Invitrogen Life Technologies). (A) Lane designations: lane 1, SeeBlue^® Plus2 (Invitrogen Life Technologies) prestained protein molecular weight standard (kDa); lane 2, adipose tissue homogenate (10 µg); lane 3, depleted adipose tissue homogenate (10 µg); and lane 4, high-abundant fraction (20 µl). (B) Lane designations: lane 1, SeeBlue Plus2 prestained protein molecular weight standard (kDa); lane 2, matched patient serum (10 µg); lane 3, depleted matched patient serum (20 µl); and lane 4, high-abundant fraction (30 µl).

Figure 9. SDS-PAGE analysis of immunoaffinity-depleted amniotic fluid patient specimen and blister fluid patient specimen

(A) Amniotic fluid patient specimen; (B) blister fluid patient specimen. Samples were resolved on a 4–12% Bis-Tris gel (Invitrogen Life Technologies, CA, USA) and stained with SimplyBlue™ Coomassie^® G-250 SafeStain (Invitrogen Life Technologies). (A) Lane designations: lane 1, amniotic fluid patient specimen (10 µg); lane 2, depleted amniotic fluid patient specimen (30 µl); and lane 3, high-abundant fraction (30 µl). (B) Lane designations: lane 1, SeeBlue^® Plus2 (Invitrogen Life Technologies) prestained protein molecular weight standard (kDa); lane 2, blister fluid patient specimen (10 µg); and lane 3, depleted blister fluid patient specimen (10 µg).