New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

Antibodies

The following antibodies or molecular probes were used at indicated concentrations for Western blot (W), immunofluorescence (IF), or FC: kindlin-2 (MAB2617 from Millipore) W: 1:1,000; talin (8D4 from Sigma-Aldrich) W: 1:1,000; paxillin (610051, clone 349 from BD Transduction Laboratories) IF: 1:500; paxillin (sc-5574 from Santa Cruz Biotechnology) IF: 1:200; integrin β1-Alexa 488-conjugated (102211 from BioLegend) FC: 1:300; integrin β1 (home-made [Azimifar et al, 2012]) W: 1:10,000, IF: 1:4,000, IP: 2 μl/mg lysate; integrin β1 (clone MB1.2 from Millipore) IF: 1:500; integrin β1 (clone JB1B from Novus Biologicals) IP: 5 μl/mg lysate; integrin α5-PE (557446 from Pharmingen) FC: 1:300; IgG2a rat isotype control (13-4321 from eBioscience) FC: 1:300; pT788/789 β1-integrin (44-872G from Thermo Fisher Scientific) W: 1:1,000, IF 1:300; pT788/789 β1-integrin (ab5189 from Abcam) W: 1:1,000, IF 1:300, phospho-threonine (clone 20H6.1 from Merck) W: 1:1,000, IF 1:500, IP: 5 μl/mg lysate; phospho-threonine (clone RM102 from Thermo Fisher Scientific) W: 1:1,000, IF 1:300, IP: 5 μl/mg lysate; PPM1F (Grimm et al, 2020) W: 1:300; SNX17 (10275-1-AP from Proteintech) W: 1:1,500.

The following secondary antibodies were used: goat anti–rabbit Alexa 488-conjugated (A11008), goat anti–mouse Alexa 488-conjugated (A11029), goat anti-rat Alexa 488-conjugated (A11006), goat anti-mouse Alexa 546-conjugated (A11003) (all from Invitrogen), FACS: 1:500, IF: 1:500; goat anti-rat HRP-conjugated (712035150) (both from Dianova) W: 1:10,000, donkey anti-rabbit Cy3-conjugated (711-165-152) (from Jakson ImmunoResearch) IF: 1:500, goat anti-mouse HRP-conjugated (172-1011) and goat anti-rabbit HRP-conjugated (172-1019) (both from Bio-Rad) W: 1:10,000.

Phalloidin-Alexa 647-conjugated (A22287 from Thermo Fisher Scientific) and DAPI (Sigma-Aldrich) were used to stain F-actin and nuclei, respectively.

Synthetic integrin cytosolic peptides

Synthetic integrin cytosolic peptides were synthesized in house by the Max Planck Institute of Biochemistry Core Facility.

β1-WT peptide (HDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK-OH), β1-scrambled peptide (EYEFEPDKVDTGAKGTKMAKNEKKFRNYTVHNIWESRKVAP-OH), β1-TT/AA peptide (HDRREFAKFEKEKMNAKWDTGENPIYKSAVAAVVNPKYEGK-OH), β1-TT/DD peptide (HDRREFAKFEKEKMNAKWDTGENPIYKSAVDDVVNPKYEGK-OH), β1-pTpT peptide (HDRREFAKFEKEKMNAKWDTGENPIYKSAVpTpTVVNPKYEGK-OH), β1-Y783A peptide (HDRREFAKFEKEKMNAKWDTGENPIAKSAVTTVVNPKYEGK-OH), β3/β5-pTpT peptide (KWDTGENPIYKEAVpTpTFTVDFTFNKFNPKYEGK-OH), and Wwc2-pTpT peptide (LLQEKGGYIPSGPIpTpTIHENEVVKSPSQPG-OH).

Plasmid construction

Point mutations in the mouse β1 integrin tail (TT788/789DD and TT788/789EE) cDNA were introduced by site-directed mutagenesis using the following primers: β1-TTDD fwd CCT ATT TAC AAG AGT GCC GTG gat gaT GTG GTC AAT CCG AAG TAT GAG G; β1-TTDD rev C CTC ATA CTT CGG ATT GAC CAC Atc atc CAC GGC ACT CTT GTA AAT AGG A; β1-TTEE fwd A AAT CCT ATT TAC AAG AGT GCC GTG gaA gaa GTG GTC AAT CCG AAG TAT GAG G; β1-TTEE rev C CTC ATA CTT CGG ATT GAC CAC ttc Ttc CAC GGC ACT CTT GTA AAT AGG ATT. For stably expressing the β1 integrin cDNAs, the cDNAs were cloned into the retroviral expression vector pLZRS. Retroviral expression constructs of β1-WT, β1-TT788/789AA, β1-Y783A, and β1-Y795A have been described (Böttcher et al, 2012). The correct sequence of all constructs was confirmed by DNA sequencing (Eurofins Genomics).

Cell lines

Fibroblasts lacking β1 integrin (β1-KO) and reconstituted with β1-WT or β1-TT788/789AA, β1-Y783A, and β1-Y795A have been described (Böttcher et al, 2012). To generate β1-TT/DD and β1-TT/EE reconstituted cell lines, the β1-KO fibroblasts were retrovirally transduced with cDNAs coding for mouse β1-TT/DD or β1-TT/EE and sorted with the flow cytometer to remove non-infected cells. UB epithelial cells were established from the metanephric kidney anlagen of E11.5 mice carrying floxed β1 integrin (Itgb1^flox/flox) alleles. The T-bud stage UBs were dissected from the metanephric kidney analgen and cultured in 3D-Matrigel in UB culture medium (DMEM/F12 supplemented with 10% charcoal/dextran treated FCS [Hyclone], 200 nM all-trans- and 9-cis retinoic acid [Sigma-Aldrich] and 15–20 ng/ml recombinant rat glial cell-derived neurotrophic factor [R&D Systems]). UB cells were immortalized with SV40 Large-T antigen in the 3D matrix before epithelial UB organoids were dissected and cultured in cell culture dishes in DMEM containing 10% FBS, 5 ng/ml glial cell-derived neurotrophic factor. The UB cells were cloned and the floxed alleles were deleted by adenoviral expression of Cre recombinase resulting in β1 integrin knockout cells (β1-KO), and reconstituted with either β1-WT, β1-TT/AA, β1-TT/DD, β1-TT/EE, β1-Y783A, or β1-Y795A. A172 sg-control and A172 PPM1F KO cells have been described (Grimm et al, 2020). All cell lines were cultured in DMEM supplemented with 10% FCS and Penicillin/Streptomycin.

Transient and stable transfection/transduction

To generate stable cell lines, VSV-G pseudotyped retroviral vectors were produced by transient transfection of 293T (human embryonic kidney) cells. Viral particles were concentrated from cell culture supernatant (Pfeifer et al, 2000) and used for infections. Cells were transiently transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

FC

FC was carried out with a LSRFortessa Flow Cytometer (BD Biosciences) equipped with FACS DiVa software (BD Biosciences). Cells were detached with trypsin/EDTA, sedimented by centrifugation (290g for 5 min) and suspended in PBS (P4417; Sigma-Aldrich) containing 1% BSA. 2–4 × 10⁵ cells were incubated with primary antibodies for 1 h on ice, washed with cold PBS supplemented with 1% BSA before incubation with the secondary antibody for 45 min on ice in the dark. Data analysis was carried out with FlowJo (version 9.4.10; BD Biosciences).

Integrin peptide ELISA

Synthetic integrin cytosolic peptides (β1-WT, β1-TT/AA, β1-TT/DD, β1-pTpT, β3/β5-pTpT, and Wwc2-pTpT) were dissolved in PBS to a concentration of 500 nM and 100 μl was immobilized on 96-well MaxiSorp plates (Nalge Nunc) overnight at 4°C. The plates were blocked with 200 μl PBS containing 3% BSA for 1 h at room temperature and then washed thrice with 200 μl PBS. Different dilutions of the anti–β1-pTpT antibodies (Thermo Fisher Scientific and Abcam) in 100 μl PBS supplemented with 3% BSA were incubated for 2 h at room temperature. After washing thrice with 200 μl PBS, 100 μl of anti-rabbit-IgG HRP-conjugate was added (1:10,000 in PBS supplemented with 3% BS) and incubated for 1 h at room temperature. The plates were washed thrice with 200 μl PBS, developed with 100 μl of ABTS substrate solution (1-Step ABTS; Thermo Fisher Scientific) before measuring absorbance at λ = 405 nm in a SpectraMax ABS Plus plate reader (Molecular Devices).

Peptide pull-down assays

Pull-down assays were performed with N-terminally desthio biotinylated β1-WT, β1-Scr, β1-TT/AA, β1-TT/DD, β1-pTpT peptides. Before use, peptides were immobilized on 25 μl Dynabeads MyOne Streptavidin C1 (10 mg/ml; Invitrogen) in Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) containing protease inhibitors (cOmplete; Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 and 3; Sigma-Aldrich) for 3 h at 4°C. Cell lysates were prepared with MPER containing protease and phosphatase inhibitors and 0.5 mg of supernatant was incubated with the peptide-coated beads at 4°C under constant rotation. For pull-downs with recombinant proteins, synthetic β1 integrin tail peptides were immobilized on 25 μl Dynabeads MyOne Streptavidin C1 (10 mg/ml; Invitrogen) for 3 h at 4°C, incubated with 2% BSA in Mammalian Protein Extraction Reagent (Thermo Scientific Scientific) for 30 min at 4°C to block unspecific binding before adding kindlin-2 or THD (conc 2.5 and 8.5 nM, respectively) and further incubation on a rotator for 2 h at 4°C. After washing the beads three times with lysis buffer, the proteins bound to the beads were eluted using Laemmli buffer, separated on a 10% SDS–PAGE, and analyzed by Western blotting.

MST

All MST measurements were performed on a Monolith NT.115 red-blue (Nanotemper) using premium-coated capillaries to reduce non-specific interaction of the proteins with the glass surface. Both interaction partners (ligand and receptor) were transferred into MST buffer (20 mM Tris, pH 7.5, 200 mM sodium chloride, 1 mM tris(2-carboxyethyl)phosphine [TCEP], 0.05% Tween-20) to avoid artifacts derived from buffer mismatches. 200 nM Atto488-labeled integrin β1-WT tails synthesized in house by the Max Planck Institute of Biochemistry Core Facility were used as ligands. The measurements were carried out at 20% light emitting diode power and 20% and 40% MST power. Data were analyzed using the MO Affinity Analysis Software (Nanotemper).

β1 integrin immunoprecipitations

For immunoprecipitation (IP) of β1 integrin, cells were lysed in β1 lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.05% sodium deoxycholate [SDC], 10 mM EDTA-containing protease inhibitors [cOmplete; Roche], phosphatase inhibitors [Phosphatase Inhibitor Cocktail 2 and 3; Sigma-Aldrich], and 10 mM phosphatase saturating substrate [para-nitrophenolphosphate [pNPP]; Sigma-Aldrich]) and incubated with rabbit anti-β1 integrin ectodomain antibodies (2 μl/mg cell lysate home-made to IP total [active and inactive] mouse β1 integrin; 5 μg/mg cell lysate JB1B to IP human β1 integrin) for 2 h at 4°C while rotating. Subsequently, lysates were incubated with 50 μl washed protein A/G Plus Agarose (Santa Cruz Biotechnology) for 2 h at 4°C. After repeated washing steps with lysis buffer, proteins were eluted from the beads using Laemmli buffer, separated on a 8% SDS–PAGE and analyzed by Western blotting.

Atomic force microscopy-based single-cell force spectroscopy

For cell attachment, tip-less V-shaped silicon nitride tip-less cantilevers (NP-O; Bruker) were plasma cleaned (PDC-32G; Harrick Plasma) and then incubated with 2 mg/ml concanavalin A (Sigma-Aldrich) in PBS at 4°C overnight. For substrate coatings, a 200-μm-thick four-segmented polydimethylsilane (PDMS) mask fused to the surface of glass bottom Petri dishes (World Precision Instruments) was used (Yu et al, 2015). Each of the four PDMS framed glass surfaces were incubated with 50 μg/ml FNIII7-10^RGD or FNIII7-10^ΔRGD in PBS at 4°C overnight. For SCFS, a Nanowizard II AFM equipped with a CellHesion module (both JPK Instruments) was mounted onto an inverted microscope (Observer Z1; Zeiss). The temperature of the medium in the Petri dish was maintained at 37°C (PetriDish heater; JPK Instruments). 200-μm-long cantilevers having nominal spring constants of 0.06 N/m were used. Each cantilever was calibrated prior the measurement by determining its sensitivity and spring constant using the thermal noise analysis using inbuild routines. To adhere a single fibroblast to the AFM cantilever, overnight serum-starved fibroblasts with confluency up to ∼80% were washed with PBS, trypsin/EDTA-detached for 2 min, suspended in SCFS media (DMEM supplemented with 20 mM Hepes) containing 1% (vol/vol) FCS, pelleted, and resuspended in serum free SCFS media. Fibroblasts were allowed to recover for at least 30 min from trypsin treatment (Schubert et al, 2014). Fibroblasts in suspension were incubated with 1 μM cilengitide (Sigma-Aldrich) for 30 min before and throughout the experiments. To attach a single fibroblast to the apex of the cantilever, the fibroblast suspension was pipetted onto the functionalized Petri dishes, the concanavalin A–coated cantilever was lowered onto a single fibroblast at 10 μm/s until a force of 5 nN was recorded and was subsequently retracted after 5 s contact at 10 μm/s for 100 μm. The cantilever-bound fibroblast was incubated for 10–15 min for binding probability experiments and ∼5 min for adhesion force measurements. Using differential interference contract (DIC) microscopy, the morphological state of the fibroblast was monitored. For binding probability experiments, the cantilever-bound fibroblast was lowered onto the substrate at 1 μm/s until a contact force of 200 pN was reached and the cantilever was retracted after 250 ms at constant hight at 1 μm/s for ≥13 μm until the fibroblast and substrate were fully separated. This resulted in a total contact time between fibroblasts and substrate of ∼300 ms. This experimental cycle was repeated for 100 times for each fibroblast, whereby the fibroblast was allowed to recover for 0.5 s between cycles and the contact area on the substrate was altered. FD curves were analyzed for unbinding events using in-build JPK software to determine binding probability as the ratio of FD curves showing unbinding events and total number of attempts. For adhesion force experiments, the rounded cantilever-bound fibroblast was lowered onto the substrate at 5 μm/s until a contact force of 1 nN was recorded. For contact times of 5, 20, 50, or 120 s, the cantilever was maintained at constant height and subsequently retracted at 5 μm/s for 100 μm until the fibroblast and substrate were fully separated. After the experimental cycle, the fibroblast was allowed to recover for a time period equal to contact time before measuring the adhesion force for a different contact time. A single fibroblast was used to probe the adhesion force for all contact times or until morphological changes, that is, spreading, was observed. The sequence of contact time measurements and area of the substrate were varied. Adhesion forces were determined after baseline correction of FD curves with JPK software (JPK Instruments).

Integrin activation assay

Cells were trypsinized, split into two fractions, and placed on ice. The cell fractions were stained for the 9EG7 epitope, reporting activated β1 integrin or total β1 integrin for 1 h on ice in PBS supplemented with 1% BSA. Cells were washed thrice with PBS and incubated with Alexa 488-conjugated secondary antibody in PBS supplemented with 1% BSA for 45 min on ice in the dark. Cells were washed and fluorescence intensity was measured by FC (BD LSRII, FACSDivaTM software; BD Biosciences).

For ELISA, starved cells were trypsinized and kept in suspension in DMEM supplemented with 3% BSA for 45 min at 37°C before seeding 40,000 cells into FN-coated (10 μg/ml) flat-bottom 96-well plates. After adhering for 40 min at 37°C, the cells were fixed with 2% PFA and washed with PBS. Afterwards, cells were blocked with PBS containing 3% BSA for 60 min at room temperature and stained for the 9EG7 epitope of β1 integrin or total integrin β1 (Millipore) overnight in PBS containing 3% BSA at 4°C. After three washes with PBS, the cells were incubated with the secondary HRP-conjugated goat anti-rat IgG antibody in PBS containing 3% BSA for 1 h at RT. Finally, cells were intensively washed, 100 μl of ABTS substrate solution (1-Step ABTS; Thermo Fisher Scientific) was added and the absorption was measured at λ = 405 nm in a SpectraMax ABS Plus plate reader (Molecular Devices).

Spreading and adhesion assays

Cells were grown to 70% confluence followed by overnight incubation in DMEM containing 0.2% FCS. After detaching with trypsin/EDTA, cells were serum starved for 1 h at 37°C in adhesion assay medium (10 mM Hepes, pH 7.4; 137 mM NaCl; 1 mM MgCl₂; 1 mM CaCl₂; 2.7 mM KCl; 4.5 g/liter Glucose; and 3% BSA [wt/vol]) or DMEM containing 3% BSA in the presence or absence of 5 μM cilengitide.

For adhesion assays, flat-bottom 96-well plates were coated with 10 μg/ml FN (Calbiochem) or 1:500 dilution of Matrigel (Corning) and blocked with PBS containing 3% BSA. Cells were then plated (40,000 per well) in adhesion buffer or DMEM containing 3% BSA ± 5 μM cilengitide and incubated for 15 or 40 min at 37°C. After vigorous washing with PBS to remove nonadherent cells, cells were fixed with PBS containing 4% PFA and adherent cells measured by staining with crystal violet (0.1% in 20% methanol) for 15 min. After extensive washing with H₂O, the color was released by 2% SDS/H₂O and the absorption was measured at λ = 570 nm in a SpectraMax ABS Plus plate reader (Molecular Devices).

For cell spreading, 40,000 cells were seeded on 10 μg/ml FN-coated coverslips, cultured in DMEM containing 3% BSA ± 5 μM cilengitide at 37°C, fixed with 4% PFA (wt/vol) in PBS, and stained with phalloidin–Alexa 488 and DAPI. At least 10 images were taken using a Zeiss LSM 780 confocal laser scanning microscope equipped with 40×/NA 1.4 oil objective and cell spreading area was measured using ImageJ (release 1.53 [Schneider et al, 2012]).

Mass spectrometry analysis

To identify proteins immunoprecipitated with anti-β1-pTpT antibodies, cells were lysed in β1 lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.05% SDC, 10 mM EDTA containing protease inhibitors [cOmplete; Roche], and phosphatase inhibitors [Phosphatase Inhibitor Cocktail 2 and 3; Sigma-Aldrich]) and incubated with rabbit anti-β1-pTpT antibodies (Thermo Fisher Scientific) for overnight at 4°C while rotating. Subsequently, lysates were incubated with 50 μl washed protein A/G Plus Agarose (Santa Cruz Biotechnology) for 2 h at 4°C. For reduction and alkylation of the proteins, previously PBS-washed beads were incubated with SDC buffer containing 1% SDC (Sigma-Aldrich), 40 mM 2-chloroacetamide (Sigma-Aldrich), 10 mM TCEP (Thermo Fisher Scientific), and 100 mM Tris, pH 8.0, at 37°C. After incubation for 20 min at 37°C, the samples were diluted 1:2 with MS-grade water (VWR). Proteins were digested overnight at 37°C with 0.5 μg trypsin (Promega). Then, the beads material was centrifuged and the supernatant was collected. In addition, the beads were washed with 100 μl of buffer A (0.1% formic acid), followed by centrifugation and collection of the supernatant. The combined supernatants were acidified with TFA (Merck) to a final concentration of 1%. Precipitated SDC was removed by centrifugation and the peptide mixture was desalted via SCX StageTips (Rappsilber et al, 2007). Samples were vacuum-dried and re-suspended in 10 μl of buffer A.

To identify phosphorylated β1 integrin, β1 integrins were immunoprecipitated from β1-WT fibroblasts kept in suspension, seeded for 45 and 90 min on FN or arrested in mitosis as indicated before. Proteins were eluted from the beads using Laemmli buffer, separated on a 8% SDS–PAGE proteins and stained with Coomassie. Gel lanes of interest were excised, chopped, and washed two times with 150 μl of destaining buffer (25 mM ammonium bicarbonate, 50% ethanol). Gel pieces were dehydrated two times in 150 μl of 100% ethanol and the gel pieces were dried by vacuum centrifugation. Then, 50 μl of digestion buffer (25 mM Tris–HCl, 10% acetonitrile, and 10 ng/μl of trypsin) was added. After incubation for 20 min on ice, 50 μl of ammonium bicarbonate buffer (25 mM) was added and the gel pieces were incubated at 37°C overnight. Peptides in the supernatant were collected and more peptides were extracted from the gel pieces by repeated incubation of the gel pieces at 25°C in 100 μl of extraction buffer (3% TFA and 30% acteonitrile), subsequent centrifugation and collection of the supernatants. Finally, the gel pieces were dehydrated by incubation at 25°C in 100 μl of 100% acetonitrile and the supernatant was unified with the supernatants from previous extraction steps. Acetonitrile was removed by vacuum-centrifugation and 70 μl of 2 M Tris–HCl as well as 10 mM TCEP and 40 mM 2-chloroacetamide was added. After incubation for 30 min at 37°C, peptides were acidified to 1% TFA and desalted using StageTips.

LC MS/MS data acquisition

For anti-β1-pTpT immunoprecipitations: Purified and desalted peptides were loaded onto a 30-cm column (inner diameter: 75 microns; packed in-house with ReproSil-Pur C18-AQ 1.9-micron beads; Dr. Maisch GmbH) via the autosampler of the Thermo Easy-nLC 1000 (Thermo Fisher Scientific). Peptides were loaded in buffer A (0.1% formic acid) at 400 nl/min and percentage of buffer B (80% acetonitrile, 0.1% formic acid) was ramped from 8% to 30% over 35 min followed by a ramp to 35% over 10 min and then 58% over the next 5 min, 95% over the next 5 min, and maintained at 95% for another 5 min.

Data acquisition on the timsTOF Pro (Brucker) was performed using timsControl. The mass spectrometer was operated in data-dependent PASEF mode with one survey thermal ionization mass spectrometry (TIMS)-MS and 10 PASEF MS/MS scans per acquisition cycle. Analysis was performed in a mass scan range from 100 to 1700 m/z and an ion mobility range from 1/K0 = 1.35 to 0.85 Vs cm⁻² using equal ion accumulation and ramp time in the dual TIMS analyzer of 100 ms each at a spectra rate of 9.43 Hz. Suitable precursor ions for MS/MS analysis were isolated in a window of 2 Th for m/z < 700 and 3 Th for m/z > 700 by rapidly switching the quadrupole position in sync with the elution of precursors from the TIMS device. The collision energy was lowered as a function of ion mobility, starting from 45 eV for 1/K0 = 1.3 Vs cm⁻² to 27 eV for 0.85 Vs cm⁻². Singly charged precursor ions were excluded with a polygon filter mask and further m/z and ion mobility information was used for “dynamic exclusion” to avoid re-sequencing of precursors that reached a “target value” of 14,500 a.u. The ion mobility dimension was calibrated linearly using three ions from the Agilent ESI LC/MS tuning mix (m/z, 1/K0: 622.0289, 0.9848 Vs cm⁻²; 922.0097 Vs cm⁻², 1.1895 Vs cm⁻²; 1221.9906 Vs cm⁻², and 1.3820 Vs cm⁻²).

To detect phosphorylated β1 integrin: Purified and desalted peptides were loaded onto a 30-cm column (inner diameter: 75 microns; packed in-house with ReproSil-Pur C18-AQ 1.9-micron beads; Dr. Maisch GmbH) via the autosampler of the Thermo Easy-nLC 1000 (Thermo Fisher Scientific) at 60°C. Using the nanoelectrospray interface, eluting peptides were directly sprayed onto the benchtop Orbitrap mass spectrometer Q Exactive HF (Thermo Fisher Scientific).

Peptides were loaded in buffer A (0.1% formic acid) at 250 nl/min and percentage of buffer B (80% acetonitrile, 0.1% formic acid) increased to 30% over 45 min followed by an increase to 60% over 5 min then 95% over the next 5 min. Percentage of buffer B was maintained at 95% for another 5 min. The mass spectrometer was operated in a data-dependent mode with survey scans from 300 to 1,650 m/z (resolution of 60,000 at m/z = 200), and up to 10 of the top precursors were selected and fragmented using higher energy collisional dissociation (HCD with a normalized collision energy of value of 28). The MS2 spectra were recorded at a resolution of 30,000 (at m/z = 200). Automatic gain control target for MS and MS2 scans were set to 3 × 10⁶ and 1 × 10⁵, respectively, within a maximum injection time of 100 and 60 ms for MS and MS2 scans, respectively. Dynamic exclusion was set to 30 ms.

LC MS/MS data analysis

Raw data were processed using the MaxQuant computational platform (version 1.6.17.0) (Cox & Mann, 2008) with standard settings applied for ion mobility data (Prianichnikov et al, 2020). Shortly, the peak list was searched against the Uniprot database of Mus musculus (55,466 entries) with an allowed precursor mass deviation of 10 ppm and an allowed fragment mass deviation of 20 ppm. To detect integrin phosphopeptides, we allowed precursor mass deviation of 4.5 ppm and an allowed fragment mass deviation of 20 ppm.

MaxQuant by default enables individual peptide mass tolerances, which was used in the search. Cysteine carbamidomethylation was set as static modification, and methionine oxidation, phosphorylation (on S, T, and Y), and N-terminal acetylation as variable modifications. The match between the run feature was enabled, and proteins were quantified across samples using the label-free quantification algorithm in MaxQuant as label-free quantification intensities.

Statistical analysis

All experiments were repeated at least three times (as indicated in figure legends). Most statistical significances (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, ns, not significant) were determined by one-way ANOVA. Two-tailed Mann–Whitney U tests were applied to determine significant differences between adhesion forces or the binding probability of fibroblast lines at different conditions, P-values comparing adhesion strengthening differences were calculated by two-tailed extra sum of squares F-test and P-values comparing rupture force difference were calculated using Kruskal–Wallis test. The statistical analysis was performed using Prism (version 9.00; GraphPad Software) in-build routines.