Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry

Abstract

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a “bird’s eye” view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0–4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.

Graphical Abstract

Mitogen-activating protein kinase (MAPK) signaling is a pathway conserved among all eukaryotes and modulates fundamental cellular processes, including cell division, proliferation, apoptosis, motility, differentiation, and metabolism. As an archetypical kinase signaling cascade, this pathway transduces signal via post-translational modifications (PTMs) (principally phosphorylation) of the components: RAS, RAF, MEK, and ERK (Figure 1A). Measurement of the modification states of these proteins, called proteoforms, should provide insight into how the pathway varies across disease states and responds to drug treatment. The most widely practiced methods for determining kinase modification state (western blotting with PTM-specific antibodies and multiple reaction monitoring of key peptides) necessitate predetermining phosphorylation sites to measure and cannot observe combinations of phosphorylations.¹ Top-down mass spectrometry (MS) offers a powerful alternative by measuring the overall stoichiometry of multiply modified proteoforms while also probing the distribution of modifications via careful analysis of fragmentation patterns. When high sequence coverage by fragment ions is achieved, even isobaric proteoforms can be discriminated as has been demonstrated with histones and in middle-down analysis of G protein-coupled receptors (GPCRs) and antibodies.^2–4 Near complete characterization is possible for analytes of this size (<15 kDa), but detailed analysis of larger proteins (>30 kDa) has remained out of reach.

Figure 1.

Overview of approach to characterizing MEK1 proteoforms. (A) The MAPK pathway propagates signaling from extracellular growth factors via a kinase phosphorylation cascade through SOS, K/H/NRAS, A/B/CRAF, MEK1/2, and ERK1/2. Regulation of this pathway occurs at multiple points but significantly at MEK1/2 where it is phosphorylated by KSR, PAK, non-RAF MAP3Ks, and ERK1/2. (B) Primary sequence of MEK1 and sites of phosphorylation within the activation segment, profile-rich domain, and C-terminal domain. (C) MEK1 was isolated from the metastatic melanoma cell line A375 and isogenic clones bearing NRAS^Q61K or MEK1^Q56P mutations via immunoprecipitation with a monoclonal antibody. Overall, phosphorylation stoichiometry was determined from ensemble MS1 measurements. (D) To determine the distribution of phosphorylations across possible modification sites, isobaric species (e.g., all diphosphorylated MEK1) were isolated by the quadrupole and fragmented by electron transfer dissociation (ETD) to produce c and z fragment ions. Fragment ions were measured by individual ion mass spectrometry (I²MS).

Two central technical limitations prevent routine analysis modes from delivering deep proteoform landscapes for mid-to-large proteoforms: accurate mass measurement of denatured intact protein ions and production/detection of large fragment ions covering the center of the sequence. For denatured protein ions, the signal-to-noise ratio decays exponentially with protein size due to the confluence of increased charges, isotopes, and chemical interference.⁵ While improved ion transmission and vacuum systems continue to increase the sensitivity of commercial mass spectrometers, these linear improvements do not match the nonlinear demands of a large proteoform analysis. The use of ion–ion reactions or photodissociation can provide higher detectability for fragmentation near the center of large protein sequences, but the high density of overlapping high-charge fragment ions makes interpretation difficult.^6,7

Individual ion mass spectrometry (I²MS) is an implementation of charge-detection mass spectrometry on an Orbitrap mass analyzer that addresses many of these challenges. Unlike typical ensemble MS, I²MS involves the simultaneous measurement of both m/z and charge of individual ions.^8,9 Ions that have too similar m/z ratios to be resolved when part of an ensemble can be resolved on their own in separate scans. Thus, I²MS is capable of measuring complex mixtures of large overlapping ions, precisely the kind of fragment ions that otherwise challenge top-down mass spectrometry.¹⁰

Largely due to BRAF hotspot mutations in over 50% of melanomas,^11–13 pharmacological inhibition of MAPK has emerged as an attractive targeted therapy, and both mutant-selective and nonselective inhibitors of multiple components have been evaluated in the clinic. While targeting MAPK has been effective in some cases, no general strategy for treating MAPK-driven cancers has emerged, and therapies that are effective are prone to acquired resistance.¹⁴ BRAF mutant-selective inhibitors (BRAFi) have been remarkably effective in the treatment of metastatic melanomas. Vemurafenib and dabrafenib selectively inhibit BRAF^V600E and can achieve dramatic tumor regression in patients harboring this mutation. Despite high response rates, recurrence of BRAFi-resistant tumors almost invariably occurs within 6 months.¹⁵ Combination treatment with MEK inhibitors delays the onset of resistance, but patients still ultimately develop resistance to such drug treatments.^16,17 Acquired resistance is frequently mediated by pathway reactivation via RAS and MEK mutation, amplification of BRAF^V600E, and PTEN loss.^18,19 MAPK reactivation also frequently occurs without a clear mechanism.¹⁴

MEK1 (UniProt Q02750, also MAP2K1) and MEK2 (UniProt P36507, also MAP2K2) are highly homologous dual-specificity kinases that canonically serve as gatekeepers of MAPK signaling, as the sole kinases that are known to phosphorylate ERK1/2. The primary structures of MEK1/2 contain an N-terminal regulatory domain, a C-terminal domain, and a protein kinase domain (Figure 1B). The pathway is not strictly linear and has negative feedback regulation via ERK activity and crosstalk from MAP3K1/2/3/8. As a result, MEK1 is phosphorylated at several sites: the activation segment, the proline-rich domain, and the C-terminus. To date, there are approximately 850 reports of PTMs on MEK1, primarily phosphorylation.²⁰ Over 60% of these reports focus on the phosphorylation of S218 and S222 (MEK1 numbering), but several other sites have been implicated in feedback regulation, mediating improved interaction with RAFs, and pathway integration.²¹ MEK1/2 also contain caspase-3 cleavage sites (D16 and D282 on MEK1 and D286 on MEK2).^22,23 Taken together, these PTMs tune the activity of MEK1 through combinations of phosphorylations, but mass spectrometry (MS) analysis of multiply modified proteins to uncover the logic of functional PTMs is a significant challenge.

Presented here is an integrated approach to produce proteoform-level information from immunoprecipitated, multiply modified proteins like MEK1. The approach uses a SampleStream device integrated directly with the mass spectrometer to automate cleaning and controlled presentation of the sample in trace amounts. In addition, individual ion mass spectrometry produces high-quality fragmentation MEK1 proteoform data for quantitative analysis to derive both PTM locations and the occupancy. The approach should find wide application in gauging drug responses in signaling pathways from models and patients.

EXPERIMENTAL SECTION

Cell Culture.

A375 and isogenic cell lines were obtained from ATCC. All cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM, Gibco 11965084) supplemented with 10% heat-inactivated fetal bovine serum (Gibco 10082147) and antibiotic–antimycotic (Gibco 15240062) at 37 °C with 5% CO₂. Sex of human cell lines: A375 (female, 54 years old). Trametinib (cat. no. HY-10999) and vemurafenib (cat. no. HY-12057) were obtained from MedChemExpress and formulated in dimethyl sulfoxide (DMSO). PAC-1 was synthesized as previously described²⁴ and formulated in DMSO.

MEK1 Enrichment and Precipitation.

MEK1 was enriched and isolated by immunoprecipitation from the metastatic melanoma cell line A375 (ATCC) with a monoclonal antibody specific for MEK1 (clone K.971.4, Invitrogen) noncovalently conjugated to magnetic beads (Figure 1C). All types of A375 cell pellets (untreated, treated and isogenic melanoma cell lines) were lysed with a freshly prepared NP-40 lysis buffer (25 mM Tris (pH 7.5), 150 mM NaCl, 1% IGEPAL CA-630 (v/v, Sigma-Aldrich I8896), 1% NP-40 substitute (v/v, Sigma-Aldrich 74385) supplemented with HALT Protease and Phosphatase Inhibitor Cocktail EDTA-free (Thermo Scientific 78441)) and was incubated at 4 °C for 30 min. The sample was then sonicated and centrifuged for 30 min at 4000 rpm. After this, the supernatant was collected. Antibody pull-downs were assembled by combining 10 mL of clarified cell lysate with 25 μL of MEK1 antibody-linked beads. After overnight incubations at 4 °C, the beads were collected, the supernatant was removed, and the beads were resuspended in 5 mL of NP-40 lysis buffer. The sample was then washed again with NP-40 lysis buffer and then washed two more times with 5 mL washes with 1× Trisbuffered saline (TBS) buffer. The samples were then transferred to a 1.5 mL low-bind Eppendorf tube and washed again twice with 1 mL of a 1× TBS buffer. Finally, the sample was washed with 1 mL of Optima-grade water. The sample was then eluted in 150 μL of 0.5% formic acid/water and mixed for 15 min at 900 rpm and 37 °C. Next, the beads were removed, and the supernatant was spun down at high speed for 10 min at 4 °C. After spinning was completed, the supernatant was collected and introduced to SampleStream (Integrated Protein Technologies, Evanston, IL) for further filtration, concentration, and buffer exchange.

SampleStream and MS Analysis.

Samples for intact measurements of MEK1 were washed and buffer exchanged individually into 70% water/30% acetonitrile/0.2% formic acid (system liquid) via SampleStream.²⁵ Specifically, samples were focused twice using 2× 250 μL of system liquid at a flow rate of 100 μL/min and then eluted in a total volume of 120 μL of system liquid at a flow rate of 40 μL/min. Samples were directly sprayed through an Ion Max Source fitted with a HESI II probe into a Q Exactive HF (Thermo Fisher Scientific). Instrumental conditions include a spray voltage of 3.3 kV, source temperature 320 °C, in-source collision-induced dissociation (CID) 15 eV, 15,000 resolving power (at 200 m/z), 700–2000 m/z, 3E6 AGC target, 50 ms maximum injection time, 10 microscans, and eFT on.

Intact MS spectra were averaged with FreeStyle (version 1.8), and deconvolution, peak picking, and integration were performed with UniDec (version 5.2.1). Phosphorylation stoichiometry figure generation and statistical tests were performed with GraphPad Prism (version 10.0.2). One-way analysis of variance (ANOVA) was performed using Dunnett’s test post hoc for multiple comparisons to the control group (wild-type (WT)/untreated cells).

All fragmentation-based data collection was subjected to I²MS analysis on the Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific) with 1 s transients. The I²MS process has been previously described in detail.^8,9,26 In short, for each acquisition, samples were loaded into a 250 μL syringe and infused via syringe pump through a custom nanoelectrospray source with a pulled borosilicate emitter at a flow rate of 300 nL/min.²⁷

Multiply modified proteoform precursor ions were isolated by the quadrupole and subsequently dissociated with higher-energy collisional dissociation (HCD) or ETD activation. Activation energies/times were set so that approximately 50% of the precursor ion remained. Injection times were manually reduced until individual fragment ions were resolved (approximately 1000 ions per scan). Individual ion processing for charge assignment was performed with STORIboard software (Proteinaceous) using an ion lifetime threshold of 300 ms, signal-to-noise threshold of 3, minimum bin size of 1, and voting probability threshold of 0.5. Fragment ion annotation was performed with a TDValidator (Proteinaceous) with manual confirmation. Fragment ion relative ratios (FIRRs) were calculated for each fragment ion by dividing its measured intensity by the sum of intensities from fragment ions with the same type and location but different degrees of phosphorylation. Approximately 1000 individual ions were multiplexed per acquisition event.

Instrumental conditions for I²MS2 on the Eclipse included eFT off, HCD pressure setting of 2 mTorr, a spray voltage of ~1.6 kV, an in-source collision-induced-dissociation value of 15 eV, and a source temperature of 300 °C. Ion signals were acquired between 350 to 2000 m/z for ~45 min using a resolution of 500,000 at 200 m/z (1 s acquisition times).

RESULTS AND DISCUSSION

MEK1 Phosphorylation Patterns, Stoichiometry, and Sites.

The proteoform landscape of MEK1 was determined by a comprehensive targeted top-down mass spectrometry approach enabled by robust immunoenrichment. The extent of MEK1 phosphorylation was determined by transferring the eluate obtained after immunoprecipitation from A375 cells into the SampleStream system for concentration, buffer exchange, and direct injection for intact MS1 ensemble measurements (Figure 1C). The baseline proteoform landscape observed for MEK1 from untreated wild-type cells shows between 0–3 phosphorylations (Figures 1C and 2A, far left). The robust pulldown of endogenous MEK1 was corroborated by western blotting (Figure S1). The localization of modifications was accomplished by isolating isobaric species and analyzing their dissociation patterns (a process depicted in Figure 1D). The ability to localize and quantify the occupancy of modification sites derives from the quality of fragmentation spectra and resulting sequence coverage (i.e., the number of backbone bonds cleaved leading to fragment ions observed and matched to a proteoform sequence). Here, we compared the measurement of fragment ions arising from collisional activation by a standard ensemble acquisition method (Figure S2A) to the new individual ion (I²MS2) approach (Figure S2B). For the unmodified form of MEK1, we found the sequence coverage for single spectra rose ~4-fold for I²MS readout (6–23%), a finding that proved critical to assign phosphorylation sites throughout this study (vide infra).

Figure 2.

Capturing MEK1 phosphorylation stoichiometry via ensemble MS measurement. (A) Intact mass spectra of MEK1 isolated from wild-type A375 following 24 h treatment with 10 μM vemurafenib, 100 nM trametinib, or 20 μM PAC-1 (from left to right, respectively). (B) Bar graph showing relative abundance of MEK1 phosphorylation states resulting from the same drug treatments as in panel (A). (C) Phosphorylation equivalents on MEK1 following drug treatment. Statistical significance is indicated (p < 0.0001) with asterisks. (D) Intact mass spectra of MEK1 isolated from isogenic A375 cell lines harboring the main genetic mutations observed in drug resistance clinically. Glutathionylated MEK1 proteoforms observed in A375-KRAS^G13D are indicated with asterisks. (E) Relative abundance of MEK1 phosphorylation states in the same isogenic A375 cell lines as in panel (D). (F) Phosphorylation equivalents of MEK1 from four isogenic A375 cell lines. Statistical significance is indicated (p < 0.0001) with asterisks.

The intact mass spectra of MEK1 from ensemble measurements showed that the extent of phosphorylation responded strongly to treatment with BRAF and MEK1 inhibitors (Figure 2A). Comparing the MEK1 from untreated A375 cells to BRAFi showed a significant decrease in phosphorylation stoichiometry, with unmodified MEK1 becoming the most abundant proteoform (Figure 2A, middle). Although the relative abundance of each phosphorylation state was measured (Figure 2B), the number of phospho-equivalents is a more convenient way to compare samples and reproducibility (Figure 2C). Treatment with vemurafenib (BRAF inhibitor) or trametinib (MEK inhibitor) decreased the phospho-equivalents from 1.3 to below 0.3 (Figure 2C). Notably, phosphorylation was not eliminated as suggested by phosphoselective western blotting (Figure S1). PAC-1, a procaspase-activating compound undergoing clinical evaluation in combination with existing therapies, slightly increased the phosphorylation level (Figure 2A). Prior work suggests PAC-1 induces MEK1 degradation by caspases as opposed to direct enzymatic inhibition.²⁸ As a result, PAC-1 did not have a reductive effect on the phosphorylation state of MEK1. This observation provides mechanistic insight for the strong synergistic effect observed for PAC-1 and BRAFi.²⁸

To evaluate the phosphorylation profile of MEK1 in BRAFi-acquired resistance, intact MS measurements were performed in genetic contexts matching those observed in the clinic. A set of four isogenic A375 cells was obtained with the following genotypes: NRAS Q61K/wild-type (NRAS^Q61K), KRAS G13D/wild-type (KRAS^G13D), MAP2K1 Q56P/wild-type (MEK1^Q56P), and wild-type/wild-type (WT). These cells were cultured, MEK1 was isolated, and the proteoform profiles were measured (Figure 2D). The relative abundance of phosphorylated proteoforms and the number of phosphor-equivalents increased to their highest levels with resistance conferring mutations (Figure 2E,F). Glutathionylation of MEK1 (+305 Da) was intermittently observed in A375 cells harboring KRAS^G13D, but the distribution of phosphorylations remained the same between these two populations of proteoforms of MEK1 (Figure 2D). The MEK1^Q56P mutation produced the most pronounced increase in phosphorylation with over twice the phospho-equivalents as wild-type. Despite MEK1^Q56P being a heterozygous mutation, no wild-type MEK1 was observed. MEK1^Q56P has exhibited some RAF-independent activity, so enhanced autophosphorylation by negative feedback relief may be the primary mechanism by which phosphorylation increases.^29–31

Phosphorylation Localization with Deep Top-Down MS.

While measurement of the overall proteoform dynamics is insightful, phosphorylation sites of MEK1 from isogenic A375 cells are associated with both activating and inhibiting kinase activity. To fully understand the activation state of MEK1, site-specific information is necessary. Ensemble tandem MS/MS measurements are frequently used to localize modifications; however, ensemble measurement of MEK1 fragment ions did not provide sufficient sequence coverage to give confident localizations (Figure S2). As is often the case for denatured proteins >40 kDa, a high number of fragment ions fall within a relatively narrow m/z window, overlap, and prevent monoisotopic mass determination. Moving to the individual ion regime evades these limitations by dramatically increasing the detectability of the high-mass fragment ions upon which PTM localization often relies.

The quality of fragment ion spectra improved with both collision- and electron-based dissociation methods, but ETD yielded more even coverage across the sequence (Figure S3). Using the I²MS acquisition modality also provided favorable sequence coverage (>70%) compared to standard ensemblemode (~7%) acquisition as demonstrated for the monophosphorylated proteoform of MEK1 from wild-type A375 cells (Figures S4–S6). This trend remained consistent for proteoforms with higher numbers of phosphorylations (Figures S7–S12). A 15 min I²MS2 acquisition was sufficient to provide >70% coverage for monophosphorylated MEK1 (Figure S6), but proteoforms carrying multiple phosphorylations required longer acquisition times to maintain similar coverage values (60 min, Figures S8 vs S9 and S11 vs S12). The fragmentation pattern of MEK1 with different phosphorylation stoichiometries was captured by I²MS and is exemplified by the triply phosphorylated form of MEK1^Q56P (Figure 3A). Extensive sequence coverage of MEK1^Q56P enabled the characterization of four phosphorylation sites of interest (S218, S222, S231, and T286, Figure S13). Although many unmodified c-ions were generated and annotated, phosphorylation was nearly exclusively identified on z ions. The number of phosphorylations on a fragment ion was correlated to fragment ion mass, and fragment ions containing more than half of the precursor mass were almost exclusively triply phosphorylated (Figure 3A,B). The fully annotated graphical fragment map containing both c and z ions is provided in Figure S13.

Figure 3.

(A) Mass spectrum of ETD fragmentation ion resulting from triphosphorylated MEK1^Q56P acquired by I²MS2. Modified fragment ion matches made by TDValidator are colored. (B) Graphical fragment map of matching z ions in panel (A) (matching c ions were observed but not shown for clarity Figure S13). (C) Intensities of z ions. (D) Fragment ion relative ratios calculated from intensities in panel (C).

The fragmentation map (Figure 3B) for I²MS2 demonstrates higher coverage in the center of the sequence compared to the termini. This apparent bias against low-mass fragment ions may arise from the S/N limitation of charge detection of individual ions; only signals above 5+ can be characterized. For denatured proteoform ions, this usually corresponds to a ~5 kDa lower boundary for fragment ion detection. However, the three lysine residues on the N-terminus enabled c-ions down to ~2 kDa to carry sufficient charge to be detected. Above this boundary, a very rich fragmentation series was recorded for unmodified and modified MEK1, encompassing multiple phosphorylation sites. When compared to the ensemble tandem mass spectra generated, the I²MS2 approach provides much deeper coverage by reducing overlapping signals and providing far more effective detection of fragment ions >10 kDa in size.

Disentangling the precise combination of triply phosphorylated MEK1 proteoforms is challenging, but their projection into the occupancies of individual phosphosites can be determined from the intensities of the pair-matched z ions (Figure 3C) and subsequently calculated relative ion ratios (Figure 3D). A stable ratio of unmodified and singly phosphorylated ions between T292 and K344 (z ions 102 to 49) indicates phosphorylation C-terminal to K344 with approximately 70% occupancy. Incomplete coverage of the C-terminal regions precludes more precise localization to specific residue(s). A distinct shift in ratios is observed at z ion index 109, which corresponds to partial phosphorylation of T292 (~50%). Two additional shifts in relative ratios were observed at z ions 172 and 177, which correspond to S222 and S218, respectively. Fragment ions N-terminal to S222 were nearly completely triply phosphorylated, indicating minimal phosphorylation between the N-terminus and S222. Taken together, these sequence-defined fragment ion relative ratios provide access to the distribution of modifications throughout the protein sequence and determine specific-site occupancies.

Site-level phosphorylation was accomplished by averaging fragment ion relative ratios in regions between the four phosphorylation sites (S218, S222, S231, and T286) plus termini and calculating the difference in phospho-equivalents between neighboring regions (Figure 4A). This analysis approach was performed for each isolated precursor and genotype (wild-type, NRAS^Q61K, and MEK1^Q56P) to provide fractional occupancy of each site (Figure 4B). By defining the phosphorylation distribution as a function of precursor modification, the order of phosphorylation can be inferred, and C-terminal phosphorylation appears to occur first, as evidenced by having the highest fractional occupancy for monophosphorylated precursors in all genotypes. The second phosphorylation event occurred at a combination of S222 and S292 with the enhanced S222 phosphorylation on MEK1^Q56P. Only by the third phosphorylation was the occupancy of S218 observed and only above 50% on MEK1^Q56P. The variance in occupancy measurements for the fourth phosphorylation was high (likely due to poor characterization of triphosphorylated fragment ions, Figure S13C) but shows further phosphorylation occupancy at S218 and S222. These observations describe a protein with the static C-terminal site and dynamic S222/S292 modification. Only with a mutation in MEK1 itself did extensive S218 and N-terminal modification occur.

Figure 4.

Analysis of fragment ion data to derive modification site occupancy. (A) Primary sequence was divided into five regions and ratios averaged. (B) Phosphorylation site occupancy as a function of genotype and isolated precursor.

Observing the interactions along the axes of genotype, modification state, and kinase activation has long been a goal of researchers, yet most studies of MEK1 have focused on singular phosphorylation sites. As described, the only way to directly capture the interaction between the mutation and PTMs on other sites is top-down mass spectrometry, which is greatly assisted by higher coverage afforded by I²MS2 analysis. Rather than simply identifying the sites of phosphorylation, the I²MS2 measurements described here define the distribution and hierarchy of the phosphorylation site usage across the entire sequence. While many reports focus on the phosphorylation of the activation segment at S218 and S222, phosphorylation of the C-terminus was disproportionately observed in monophosphorylated MEK1 across all genotypes studied here. The static nature of this modification, even in otherwise highly phosphorylated species, may indicate a basal level of negative feedback from ERK2. In contrast, phosphorylation of the proline-rich domain (represented by T286) increased proportionally to overall phosphorylation, which represents dynamic downregulation of MEK1 activity by ERK1 and PAK. Further, phosphorylation of S218 was a rare event and only observed when the number of phospho-equivalents reached three or more.

Comparing across genotypes, MEK1 derived from wild-type and NRAS^Q61K cells had similar distributions of phosphorylation, while MEK1^Q56P displayed a highly aberrant phosphorylation pattern (Figure 4B, at left). In this context, S218 phosphorylation achieved its highest extent. Although the precise mechanism by which MEK1^Q56P confers resistance remains unclear, some experiments point to decoupling of the ERK-mediated negative feedback. As phosphorylation of the C-terminus and proline-rich domain remained relatively consistent with other genotypes, these data suggest feedback loss may arise from loss of allostery.

Mutations in KRAS, NRAS, and MEK1 critically impact melanoma patient outcomes through MAPK pathway reactivation. Targeted structural characterization in these mutational contexts is crucial for understanding the recurrence of myeloma and provides a view into the mechanisms by which melanoma evades BRAF and MEK inhibitor therapy. Our tiered approach of determining intact phospho-landscapes followed by subsequent precise site localization gains additional in-depth insight into proteoforms and their relation to function,³² such as higher levels of phosphorylation on MEK1 mutants despite evidence of strong negative feedback regulation.

CONCLUSIONS

In the improved readout of proteoform fragmentation spectra, we used I²MS for precise localization of PTMs and quantification of their occupancies using fragment ion relative ratios.³³ This kind of information would be extremely difficult to obtain with ensemble detection of ETD fragmentation spectra, given its bias for low-mass ions.¹⁰ Importantly, the specific approach used here may be extensible from a protein of moderate size (43 kDa) to other endogenous proteins approaching 100 kDa.

The phosphoprotein patterns determined from the integrated approach established here provide a new way to quantify the extent of signaling through the MAPK pathway. These patterns can striate candidate treatments and find unexpected PTMs by virtue of the full sequence coverage afforded by top-down MS. In addition, the immunoproteoform assay format is translatable to clinical specimens with minimal adjustment. By providing a simple, “bird’s eye” view of the stoichiometry and hierarchy of modification site usage on MEK1, we forward a new level of capability to capture the dynamic PTM logic of endogenous oncoproteoforms. Overall, the proteoform signatures align well with cellular phenotypes, but future work will determine if such profiles are reflected in clinical samples of melanoma patients.