From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography—tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and –activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and -activating properties, occurs in different NH 2 -terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.

INTRODUCTION
Neutrophils are the most abundant leukocyte type in human blood (1). These innate immune cells are endowed with a comprehensive anti-microbial machinery and are usually the first responders to infection or tissue injury (2). Adequate spatiotemporal trafficking and activation of neutrophils are essential to guarantee immune surveillance and to prevent inappropriate immune activation leading to tissue damage. Chemokines play a central role in this process (3). These low molecular mass proteins function primarily by activation of dedicated heptahelical G proteincoupled receptors (GPCRs) (4,5). However, the precise chemokine activity and availability in vivo are the labyrinthine outcome of multiple regulatory mechanisms with a significant role for posttranslational modifications (6)(7)(8)(9). Mature secreted chemokines are susceptible to site-specific proteolysis, citrullination, nitration and glycosylation, with modificationand ligand-dependent consequences for their biological functions [reviewed in (6,(9)(10)(11)].
CXCL8 or interleukin (IL-) 8 is the most powerful neutrophilattracting chemokine in humans. CXCL8 is a small protein without glycosylation (aglycosyl) and is produced by virtually any cell type upon appropriate stimulation. It has been widely implicated in diseases including autoimmune disorders and cancer (12,13). CXCL8 was described for the first time in the late 80s by four independent research groups (14)(15)(16)(17)(18). Upon its discovery, it was clear that CXCL8 displays a remarkable degree of natural NH 2 -terminal sequence heterogeneity, due to its sensitivity to proteolytic modification in particular (15,19). Follow-up research revealed that, in general, NH 2 -terminally shortened CXCL8 proteoforms exhibit a superior capacity to chemo-attract and activate neutrophils (up to 30 times more potent) as compared to full-length CXCL8(1-77) on condition that the conserved ELR motif remains intact (20)(21)(22)(23)(24)(25)(26). In addition, elongated CXCL8(-2-77)-that presumably originates from alternative splicing of the signal peptide-has a moderately increased biological activity as compared to CXCL8(1-77), but is less efficiently processed to the more potent form CXCL8(6-77) (27). Enzymes responsible for CXCL8 cleavage include plasmin, thrombin, CD13, matrix metalloproteinase (MMP)-1, MMP-8, MMP-9, MMP-13, MMP-14 and cathepsins B, G, K, L, and S. They are usually upregulated in pathological conditions, supporting the idea that CXCL8 processing becomes predominantly relevant during inflammation. This further sparked our interest to develop methods sensitive enough to quantify CXCL8 proteoforms in small-sized human samples.
The currently available standard immunoassays do not discriminate between authentic and processed chemokines with divergent biological activities. Indeed, most commercially available antibodies recognize the different forms of a specific chemokine with similar efficiency. Western blot approaches may point toward modifications with major effects on the molecular mass of the protein involved, but cannot detect subtle proteolysis nor reveal the exact identity and activation state of the processed molecule. Moreover, physical separation of chemokine forms is challenging since proteins with minimal structural differences co-elute from conventional columns used in ion exchange and reversed phase (RP) chromatography (20).

Patients
Patients were recruited at the University Hospital Leuven after informed consent according to the ethical guidelines of the Declaration of Helsinki. Parents or legal guardians signed the informed consent on behalf of children. Synovial fluid was collected only if joint aspiration was required for treatment. Synovial fluids were collected in BD vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (BD Biosciences, East Rutherford, NJ). The Ethics Committee of the University Hospital Leuven approved experiments involving human samples (S59874 and ML1814).

Immunosorbent Isolation of CXCL8 From Synovial Fluids
For each sample, 5 µg biotinylated polyclonal rabbit anti-human CXCL8 (vide supra) was coupled to 25 µl streptavidin-coated magnetic particles (Dynabeads TM M-280 Streptavidin, Thermo Scientific, Waltham, MA) during 30 min at room temperature (RT) under continuous rotation. Antibody-labeled beads were washed four times with 0.5 ml PBS using a DynaMag 2 magnet (Thermo-Scientific) and incubated with 0.5 ml synovial fluid or with 0.5 ml human plasma enriched with CXCL8(1-77), CXCL8(6-77) and CXCL8(9-77) (5 ng of each form). After 30 min of incubation at room temperature under rotation, beads were washed four times with 0.5 ml PBS and CXCL8 proteoforms were eluted with 0.1 M glycine pH 2.8 (elution volume of 20 µl). Samples were directly loaded in a cooled autosampler (5 • C) and analyzed by nano-LC-MS/MS. To investigate the kinetics of CXCL8 processing in the presence of synovial fluids, exogenous CXCL8(1-77) (500 ng) was subjected to human synovial fluid (20 µL) for a period of 0, 3, 6, 12 or 20 h and the same procedure was followed.

Production of Natural CXCL8 by Osteosarcoma Cells (MG-63)
The osteosarcoma cell line MG-63 was grown in Eagle's minimum essential medium and stimulated with the synthetic double stranded RNA poly rI:rC (50 µg/ml; P-L Biochemicals, Milwaukee, WI) to produce CXCL8 as described (29). To isolate CXCL8 variants, MG-63 cell culture supernatant was concentrated and partially purified using controlled pore glass (CPG) and heparin affinity chromatography (GE Healthcare; Chicago, IL). Protein elution was achieved using a NaCl gradient of 0.05-2.0 M in 50 mM Tris-HCl (pH 7.4). Heparin-Sepharose fractions containing CXCL8 immunoreactivity, demonstrated by a specific CXCL8 ELISA developed in our laboratory (20), were further purified on a C8 Aquapore RP-300 column (220

Development of a Nano-LC-MS/MS-Based Tool for Identification of CXCL8 Proteoforms
Tandem mass spectrometry (MS/MS) has become the standard technique for protein identification in complex biological samples. Given its precision, accuracy and selectivity, we selected nano-LC-MS/MS as the method of choice for development of a novel tool that allows for identification and quantification of CXCL8 proteoforms in complex smallsized samples without the use of proteases (e.g., trypsin) during sample preparation. Authentic CXCL8(1-77), elongated CXCL8(-2-77) and all six naturally occurring truncated CXCL8 proteoforms i.e., CXCL8(2-77), CXCL8(3-77), CXCL8(6-77), CXCL8(7-77), CXCL8(8-77), and CXCL8(9-77) were chemically synthesized and used as reference molecules (protein sequences of CXCL8 forms are provided in Supplementary Figure 1). For optimization of nano-LC-MS/MS parameters, we took advantage of the fact that CXCL8 proteins contain one Asp-Pro (-D-P-) bond, i.e., the peptide bond with the highest sensitivity to acidic hydrolysis (Figure 2A; Supplementary Figure 1). To guarantee maximal sensitivity of the method, MS/MS parameters were optimized to ensure that only the D-P bond breaks during top-down CID (Figure 2A). This results in the generation of two signature fragment ions and, importantly, a minor loss in intensity. The rationale for this approach, as exemplified for full-length CXCL8(1-77), is shown in Figure 2A.
The different CXCL8 proteoforms share a common, COOHterminal fragment ion (m/z value 615.3 with 4 charges)that can be used as an internal control-and have specific NH 2 -terminal fragment ions. The combination of detection of signature fragment ions, generated by fragmentation of a pre-selected precursor ion, at the expected elution time point, strongly indicates the presence of a specific CXCL8 proteoform. To explore whether nano-LC-MS/MS can be used for quantification of CXCL8 proteoforms, mixtures were prepared containing equal amounts of CXCL8(1-77), CXCL8(6-77), and CXCL8(9-77). We found that CXCL8 proteins can   be quantified by determining the intensity of their fragment ions in the extracted ion chromatogram (EIC), that shows the detection of signature fragment ions generated by fragmentation of a specific precursor ion during protein elution. Doseresponse curves demonstrating the simultaneous quantification of 3.1 pg−12.5 ng CXCL8(1-77), CXCL8(6-77), and CXCL8(9-77) in MRM mode are shown in Figure 2D. Up to five CXCL8 proteoforms can be measured simultaneously in MRM mode without loss of sensitivity. Analysis of a mixture containing CXCL8(1-77) and CXCL8(6-77) (ratio 1:4) confirmed succesful quantification if samples contain unequal amounts of CXCL8 proteoforms (Supplementary Figure 2). Finally, the dynamic range of detection was at least four orders of magnitude ( Figure 2D).

DISCUSSION
The significance of posttranslational modifications has been evidenced in various immunological settings. For example, caspase-mediated proteolytic activation is a prerequisite for generation of biologically active IL-1β and IL-18 (40,41). Protein citrullination, presumably, plays an important role in the pathogenesis of multiple sclerosis and RA (42)(43)(44). Intense former research efforts demonstrated the potentially substantial effects of posttranslational modifications on the biological activity and receptor preferences of chemokines in vitro and in vivo [reviewed in (6,10)]. However, their importance in patient samples is unclear due to a lack of specific detection methods for proteoforms with different activities. In the past, we purified multiple naturally processed chemokines from cell culture supernatants using a multi-step analytical purification method that combines (1) adsorption to CPG, (2) antibody or heparin-Sepharose affinity chromatography, (3) cation exchange chromatography and (4) RP-HPLC and confirmed protein identity by Edman degradation (20,33). Though proven successful, this time-consuming analytical approach requires liters of starting material. The lack of more straightforward immuno-assays able to distinguish differentially processed chemokines and the limited volumes of clinical samples available for research purposes hamper detection of distinct chemokine forms in human body fluids. Despite the difficulties that challenge detection and quantification of chemokine proteoforms with different activities in biological samples, a few scientific reports validating the occurrence of differentially processed chemokines in vivo were published. Consequently, the role of posttranslational modifications in fine-tuning the precise chemokine activity can no longer be neglected. Proteoforms of CXCL10 and CXCL12 that lack two NH 2 -terminal residues as a result of CD26mediated proteolysis, have been detected in human plasma (45,46). Moreover, an emerging body of evidence supports the notion that interfering with chemokine processing may have therapeutic benefits. For example, in vivo inhibition of the protease CD26 resulted in elevated concentrations of intact CXCL10 accompanied by enhanced anti-tumor activity in mice (7). Likewise, inhibition of CD26 results in enhanced availability of biologically active CXCL12, thereby improving wound healing (8). Inhibition of the isoenzyme of glutaminyl cyclase that is responsible for cyclization of glutamine into pyroglutamic acid, coincided with reduction of CCL2-driven monocyte recruitment in vivo with beneficial effects on atherosclerosis progression (47).
In the present study, we introduce in an exemplary way a straightforward, approach for simultaneous detection and relative quantification of differentially processed proteoforms of the aglycosyl chemokine CXCL8. Following immunosorbent sample preparation, top-down ion trap tandem mass spectrometry with limited fragmentation energy is exploited for identification of native CXCL8(1-77), elongated CXCL8(-2-77), and naturally occurring truncated CXCL8 proteoforms lacking one to eight NH 2 -terminal amino acids. Considering that we aimed to detect specific proteoforms with minimal structural differences, a major advantage of our top-down approach over commercially available bottom-up specific enrichment methods such as "stable isotope standards and capture by anti-peptide antibodies" (SISCAPA) is the fact that there is no requirement for initial enzymatic protein digestion and/or stable-isotope labeling (48). Indeed, protein digestion by proteases such as trypsin, the preferred enzyme in bottom-up proteomics, will cleave NH 2 -terminal peptides from chemokines and prevent correct relative quantification of NH 2 -terminal proteoforms known to have different activities. It is generally acknowledged that information on the occurrence of specific proteoforms of the molecule of interest gets lost easily upon protein digestion used in bottom-up proteomics (49,50). Furthermore, anti-peptide specific antibodies cannot be used to discriminate between chemokine proteoforms. Although marking peptides with stable isotopes may improve the sensitivity of mass spectrometry-based methods for protein quantification and reduces experimental bias, working with stable isotope-labeling is less practical, implies a higher cost and the need of larger amounts of starting material as compared to label-free approaches (51). Moreover, sample preparation and data analysis are more complex.
In contrast to mass spectrometric immunoassays (MSIA), the ISTAMPA procedure can be performed with a standard electrospray ionization -ion trap mass spectrometer. Therefore, this method is accessible to any laboratory well equipped for standard tandem mass spectrometry. The generation of only two signature fragment ions using limited fragmentation energy ensures maximal sensitivity of the method. Our method was validated in patient samples and we confirmed the existence of natural NH 2 -terminally cleaved CXCL8 proteoforms in synovial fluids from arthritis patients. Although oligoarticular/polyarticular JIA and RA were traditionally considered to be antigen-driven autoimmune diseases with a predominant role for T cells, the complete spectrum of immuneinflammatory responses as seen in patients probably depends on complex interactions between innate and adaptive immunity. An important role for neutrophils, acting at the crossroads of innate and adaptive immunity, in synovial inflammation characteristic of JIA and RA has been speculated (35,36,38,39,52). Detection of CXCL8 proteoforms with superior potencies in synovial fluids from patients favors this idea and opens a window of opportunity for identification of potential drug targets. Importantly, neutrophils may promote their own recruitment and activation by secreting enzymes known to process CXCL8 into more potent, truncated proteoforms to set up an auto-amplification loop of neutrophilic inflammation, eventually leading to tissue damage if patients are not treated appropriately (24).

DATA AVAILABILITY STATEMENT
The original data presented in this study are publicly available. Data can be found here: [ftp://massive.ucsd.edu/ MSV000086840/]. Other data are included in the article or Supplementary Materials.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of the University Hospital Leuven. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
BN, PV, LD, and CW were responsible for diagnosis and recruitment of patients. MM, SA, JVa, AM, RJ, SV, EG, EM, GO, and PP performed experiments and analyzed data. PP supervised the study. MM wrote the initial manuscript with assistance of SA. All authors contributed to the study conception and design, commented on previous versions of the manuscript and approved the final version of the manuscript.