Tip based fractionation of batch-enriched phosphopeptides facilitates

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Tip based fractionation of batch-enriched phosphopeptides facilitates easy and robust phosphoproteome analysis. Alireza Dehghani, Markus Gödderz, and Dominic Winter J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00256 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017

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Journal of Proteome Research

Tip based fractionation of batch-enriched phosphopeptides facilitates easy and robust phosphoproteome analysis. Alireza Dehghani1, Markus Goedderz1, Dominic Winter1* 1 Institute for Biochemistry and Molecular Biology, University of Bonn, Germany

ABSTRACT: The identification of large numbers of phosphopeptides from complex samples largely relies on sample fractionation in order to reduce complexity and to allow using large amounts of starting material. For such experiments, commonly fractionation of whole cell lysate digests followed by enrichment of phosphopeptides from the single fractions is performed. We evaluated the tip-based fractionation of batch-enriched phosphopeptides as an alternative method. We compared three tip-based fractionation methods employing strong cation exchange (SCX), strong anion exchange (SAX) and C18 material for basic reversed phase (BRP) fractionation using HeLa whole cell lysate digests. We show that SCX tips are superior to BRP and SAX tips due to a more efficient retention and distribution of phosphopeptides as well as a better resolution. Furthermore, we show that tip based fractionation results in a similar performance as fractionation followed by phosphopeptide enrichment of the single fractions and outperforms analysis of unfractionated phosphopeptide enriched samples with long chromatography gradients. Our fractionation approach using SCX tips is straightforward,

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reproducible and requires a fraction of time, effort and instrumentation compared to the fractionation of whole cell lysate digests with subsequent enrichment of phosphopeptides from the single fractions.

KEYWORDS: Large scale phosphoproteomics, Tip based fractionation, Phosphopeptides, Phosphorylation, Liquid chromatography, Mass spectrometry, SAX, SCX, BRP

Introduction Protein phosphorylation plays an important role in a multitude of cellular functions such as signal transduction, regulation of enzyme activity and protein-protein interactions.1 Since the first development of workflows for large-scale phosphoproteome analysis using mass spectrometry, a great variety of protocols for the enrichment and fractionation of phosphopeptides has been established.2,3 This led to the identification and quantification of phosphorylation sites in a plethora of cells and tissues providing valuable information for the identification of cellular mechanisms.4 The development of these workflows was motivated by certain challenges preventing the identification of phosphopeptides in complex peptide mixtures resulting from the proteolytic digestion of whole proteomes. The most important to mention are low abundance and altered detection efficiencies compared to non-phosphorylated peptides in the sample. The latter can be due to altered ionization of phosphorylated peptides as well as loss of phosphopeptides caused by unspecific binding to metal ions on reversed phase columns.5–7 To enhance detection of phosphopeptides, several methods have been established for their enrichment based on the negatively charged phosphorylation group. These methods can be grouped into three categories: i) Immobilized Metal Ion Affinity Chromatography (IMAC), ii)


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Metal Oxide Affinity Chromatography (MOAC), and iii) immunoaffinity chromatography.2,3,8,9 Beside these affinity methods, liquid chromatography (LC) based approaches have been used for separating phosphopeptides from their non-phosphorylated counterparts. The most popular ones are ion exchange chromatography (SAX10 and SCX11), hydrophilic interaction liquid chromatography (HILIC)12, and electrostatic repulsion-hydrophilic interaction chromatography (ERLIC).13 Similar to IMAC and MOAC, the negative charge of the phosphate group allows for separation of phosphopeptides from their non-phosphorylated counterparts in these approaches.2 Although the mentioned chromatographic separations can be used as a stand-alone method for phosphopeptide enrichment in large-scale phosphoproteomics studies, the combination of peptide fractionation (usually by SCX, SAX, or high-pH reversed-phase chromatography) with an enrichment for phosphopeptides by IMAC or MOAC are most common.2,3 While sample fractionation can principally be performed either before or after phosphopeptide enrichment, the vast majority of studies employed fractionation followed by enrichment of phosphopeptides for each fraction.2,3 These approaches are very laborious and time-consuming since each of the generated fractions has to be processed individually. Therefore, enrichment of phosphopeptides prior to fractionation would be more cost effective and less time-consuming, as for each sample only a single phosphopeptide enrichment has to be performed. Furthermore, it would be possible to miniaturize fractionation equipment as the samples to be fractionated are several micrograms of phosphopeptides rather than several milligrams of whole cell lysate digests. To our knowledge, so far only a handful of studies have been published performing fractionation of phosphopeptides after their enrichment. Engholm-Keller et al. developed a workflow which consists of SIMAC14 enrichment of phosphopeptides followed by their fractionation by HILIC (TiSH workflow)15 while Wiśniewski et al.performed filter aided sample preparation (FASP) and


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TiO2 enrichment prior to phosphopeptide fractionation using SAX tip-based columns.16 Batchenrichment of phosphopeptides using TiO2 followed by TMT labeling and high-pH reversedphase fractionation was first used by Erickson et al.17 and later by Possemato et al.18 while Iesmantavicius et al. was the first to use SCX tip-based fractionation after batch enrichment of phosphopeptides using TiO2.19 Furthermore, during the preparation of this manuscript, an alternative fractionation strategy by SCX tips of IMAC batch-enriched phosphopeptides was published.20Also for samples enriched for other PTMs SCX tip based fractionation has been shown to be successful. Weinert et al.21, for example, used SCX tips to fractionate succinylated peptides while Iesmantavicius et al. employed this strategy for ubiquitinated peptides.19 Tipbased fractionation of peptides follows the principle of STAGE tips which are micro columns manufactured using chromatographic beads immobilized in a teflon meshwork.22 In this context, the advantages of this method over conventional chromatographic methods are for example their low cost, ease-of-use, reproducibility, and flexibility. For the fractionation of peptide samples in general, tip-based columns are by now well established, especially in the format of strong anion exchange tips (e.g. 23,24). In this study, we compared methods for the analysis of batch-enriched phosphopeptide samples. This includes analysis of unfractionated samples using long gradients, fractionation by tip-based SAX, SCX and BRP columns as well as the commonly used setup using a high capacity SCX column in combination with a chromatography system.

Experimental Section Cell culture, lysis and protein digestion


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HeLa cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% FCS, 100 µg/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine at 37° C and 5% CO2. Cells were washed twice with ice-cold PBS, harvested using a cell scraper and snapfrozen using liquid nitrogen. Samples were lyzed in buffer containing 8 M urea and the protein concentration determined using the DC protein assay (Bio-Rad, Hercules, CA, USA). Proteins were reduced using either 5 mM dithiothreitol (DTT) or 10 mM β-mercaptoethanol (β-ME) at 56° C, 800 rpm for 25 min and alkylated for 30 min in either 20 mM acrylamide or 14 mM iodoacetamide in the dark. The reaction was quenched with the same amount of reducing reagent (β-ME or DTT) at room temperature (RT), 800 rpm for 15 min, samples diluted 1:5 by 25 mM TRIS/HCl (pH 8.2) and CaCl2 was added to a final concentration of 1 mM followed by trypsin at an enzyme-to-substrate ratio of 1:200. Proteins were digested overnight at 37° C, peptides purified using Oasis HLB cartridges (Waters, Milford, MA, USA) and the eluate fractions dried in a vacuum centrifuge. Samples with protein contents of up to 1 mg were purified with 10 mg cartridges, samples with higher amounts using 400 mg cartridges. Cartridges were initially equilibrated using acetonitrile (ACN) followed by 50% ACN/0.5% acetic acid (AcOH) and 0.1% trifluoroacetic acid (TFA). Dried samples were resuspended using 0.1% TFA, loaded to the column, washed using 0.1% TFA, eluted using 50% ACN 0.5% AcOH and dried again using a vacuum centrifuge. Phosphopeptide enrichment Dried peptides were resuspended in 500 µl 80% ACN, 5% TFA, 1 M glycolic acid.25 TiO2 (Sachtopore-NP TiO2, 5 µm, 300 Å, Sachtleben, Duisburg, Germany) was added at a ratio of 1:6 (peptides to beads) followed by incubation at RT, 1200 rpm for 15 min. Beads were pelleted by centrifugation at 13,000 × g for 1 min and the supernatant discarded. Subsequently, TiO2 beads


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were transferred to new tubes using 1 ml of 80% ACN, 1% TFA, centrifuged, and the supernatant discarded. After washing the beads with 1 ml of 20% ACN, 0.1% TFA they were centrifuged, the supernatant discarded and dried using a vacuum centrifuge. Phosphopeptides were eluted from TiO2 beads by adding 200 µl 1% NH4OH and incubation at RT, 1200 rpm for 15 min. Beads were pelleted by centrifugation and supernatants transferred to new tubes followed by acidification using 10 µl formic acid (FA). Peptides were dried using a vacuum centrifuge and resolubilized in 500 µl 70% ACN, 0.1% TFA. TiO2 was added again to each sample at a ratio of 1:6 (peptides to beads) and the beads were washed with 50% ACN, 0.1% TFA. Phosphopeptides were eluted by incubation with 200 µl 1% NH4OH at RT, 1200 rpm for 15 min, acidified using FA and desalted with 10 mg Oasis HLB cartridges. The resulting elution fractions were dried using a vacuum centrifuge and stored at -20°C. Strong Cation Exchange (SCX) Chromatography Separation of peptides using SCX chromatography was performed on an Äkta Purifier system (GE Healthcare, Little Chalfont, United Kingdom) using a 100 × 9.4 mm Polysulfoethyl A column (PolyLC Inc., Columbia, MD, USA) as described initially by Villen et al.25 The following solvents were used: solvent A: 7 mM KH2PO4, pH 2.65 and 30% ACN; solvent B: 7 mM KH2PO4, pH 2.65, 30% ACN, 350 mM KCl and solvent C: 50 mM K2HPO4, pH 7.5, 500 mM NaCl. pH values were adjusted using phosphoric acid (H3PO4) before addition of ACN. The flow rate was set to 2 ml/min and fractionation was performed at 8°C. The column was equilibrated using solvent C and A as described by Villen et al.25 and peptides loaded in 500 µl of solvent A followed by a washing step of 4 min. Fractionation was performed with a linear gradient from 100% solvent A to 70% solvent A/30% solvent B in 48 min followed by a linear gradient to 100% solvent B in 2 min, 2 min at 100% solvent B and 8min of water. Across the


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gradient, 12 fractions of 12 ml each were collected for the first experiment and 6 fractions of 24 ml each for comparison to the tip based columns. Each fraction was lyophilized and peptides were desalted using Oasis cartridges followed by TiO2 enrichment (fractionation of whole cell lysates). Pipette tip-based strong cation-exchange (SCX) fractionation of phosphopeptides Six elution buffers containing 7 mM KH2PO4 and 30% ACN were prepared with ascending concentrations of KCl (0 mM, 30 mM, 60 mM, 90 mM, 120 mM, and 350 mM) and pH values adjusted to 2.65 using H3PO4. 12 disks of Empore Cation-SR material (3M, St. Paul, MN, USA) were placed into 200 µl micropipette tips. Tip-columns were equilibrated by sequential application of 100 µl of methanol, elution buffer 6 (7 mM KH2PO4, 30% ACN, and 350 mM KCl), water, equilibration buffer (50 mM K2HPO4, pH 7.5, 500 mM NaCl), water and elution buffer 1 (7 mM KH2PO4, 30% ACN), respectively, followed by centrifugation at RT, 2500 × g for 3 min each. Samples were solubilized in 200 µl elution buffer 1 and loaded on the equilibrated tip-columns followed by centrifugation at RT, 2500 × g for 3 min. The flow-through was collected as the first fraction and 5 more fractions were collected using 200 µl of elution buffers 2 to 6 followed by centrifugation at RT, 2500 × g for 3 min. All fractions were dried using a vacuum centrifuge. Pipette tip-based strong anion-exchange (SAX) fractionation of phosphopeptides SAX fractionation was adapted from Wiesniwski et al.23 Tip-columns were manufactured using 12 disks of Empore Anion-SR material (3M, St. Paul, MN, USA). SAX buffers containing 20 mM AcOH, 20 mM H3PO4, and 20 mM boric acid were prepared with descending pH values (pH 11, 8, 6, 5, 4, 3) by the addition of NaOH. To the final elution buffer (pH3) 0.25 M NaCl was


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added. Tip-columns were washed and equilibrated sequentially by addition of 100 µl of MeOH, 1 M NaOH, and loading buffer (pH 11) followed by centrifugation at RT, 2500 × g for 3 min. Phosphosamples were resuspended in 200 µl of loading buffer and loaded onto the column followed by centrifugation at RT, 2500 × g for 3 min. The flow-through was collected as the first fraction followed by five subsequent fractions eluted using 200µl of buffer with descending pH value by centrifugation at RT, 2500 × g for 3 min each. All samples were dried using a vacuum centrifuge. Pipette tip-based concatenated high-pH reversed-phase (Basic-RP) fractionation We followed the strategy described by Han et al.26 with several changes concerning the type of reversed phase material used and the percentage of acetonitrile for elution of peptides in the single fractions. Tip-columns were prepared using 12 disks of C18 Empore Extraction material (3M, St. Paul, MN, USA). Buffers with 10 mM ammonium formate and increasing amounts of ACN were prepared for phosphopeptide elution and their pH value adjusted to pH 10 by the addition of 28% NH4OH. Columns were equilibrated by sequential application of 100 µl of the following solutions and centrifugation at RT, 2500 × g for 1 min: MeOH, 10 mM ammonium formate, 50% ACN and 10 mM ammonium formate, 2% ACN. Samples were solubilized in 200 µl of 10 mM ammonium formate 2% ACN and the flow-through was collected as the first fraction. Subsequent fractions were eluted using 200 µl of ammonium formate buffers with 6%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 33%, 36%, 40%, 50%, 60%, 70%, 80% and 90% ACN. Finally, tip-columns were eluted using 100% ACN. Later, these eluates were combined to form 6 fractions in the following way: fraction 1 consisted of eluates with 6%, 18%, 28%, and 50% ACN; fraction 2 of eluates with 10%, 20%, 30%, and 60% ACN; fraction 3 of eluates with 12%, 22%, 33%, and 70% ACN; fraction 4 of eluates with 14%, 24%,


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36%, and 80% ACN; fraction 5 of eluates with 16%, 26%, 40% and 90% ACN; and fraction 6 was the combination of the flow-through of the sample loading step and the last eluate fraction (100% ACN). After sample pooling, all fractions were dried using a vacuum centrifuge. LC-MS/MS data acquisition All samples were desalted using C18 STAGE tips as described elsewhere27and dried using a vacuum centrifuge. Desalted samples were resuspended in 5% ACN, 5% FA solution and loaded on in-house manufactured analytical columns at a flow rate of 1 µl/min solvent C (0.1% FA in water) using a nanoflow UHPLC system (EASY-nLC 1000, Thermo Fisher Scientific, Waltham, MA, USA). Columns were prepared by packing a 100 µm ID fused silica spray tip pulled with a P2000 laser puller (Sutter Instruments, Novato, CA, USA) with 5 µm ReproSil-Pur 120 C18-AQ particles (Dr. Maisch, Ammerbuch-Entringen, Germany). Columns were equilibrated with several injections of 50 mM citrate before sample loading.6 Separation was performed at a flow rate of 400 nl/min with 60 or 90 min gradients from 99% solvent A (water with 5% DMSO28, 0.1% FA) 1% solvent B (ACN with 5% DMSO, 0.1% FA) to 65% solvent A 35% solvent B. 240 min segmented gradients were used for the analyses of unfractionated samples. Segmented gradients consisted of a 200 min linear gradient from 1% to 25% solvent B, followed by a 30 min step to 30% solvent B and a 10 min step to 35% solvent B at 400 nl/min. Peptides eluting from the column were ionized at 1.6 kV in the positive ion mode in the nanosource of an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Survey scans were acquired in the Orbitrap analyzer from m/z 400 to 1200 at a resolution of 30,000 followed by fragmentation of the 10 most abundant ions in the LTQ part of the instrument. Singly charged ions and such with unassigned charge states were excluded from


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MS/MS fragmentation. The repeat count was set to 1 and dynamic exclusion to 30s for all gradients. Multi-Stage Activation (MSA)29 was activated triggered by neutral-losses of m/z 24.5, 32.7, 49, and 98. Data analysis Generated *.raw files were analyzed using MaxQuant v.1.5.0.0.25. For each replicate, single fractions (1, 6 or 12) were analyzed in one combined search. The database used was SwissProt (release 2015_04, 20192 entries). Fixed modifications were set to carbamidomethyl (in the case of alkylation with iodoacetamide), or propionamide (in the case of alkylation by acrylamide) at cysteine residues. Variable modifications were as follows: acetylation at protein N-termini, oxidation at methionine, carbamylation at lysine and peptide N-termini, as well as phosphorylation at serine, threonine, and tyrosine. Matching between runs was used with default settings. The minimal score for modified peptides was set to 40 and the minimal delta score for modified peptides to 9. Gene ontology analyses were performed using PANTHER (www.pantherdb.org) using the “PANTER Overrepresentation Test” (release date 2017/04/13) in combination with the GO Ontology database (release date 2017/09/26).30,31

Results and Discussion Fractionation of batch-enriched peptides by SCX chromatography leads to substantial loss of phosphopeptides The goal of this study was to establish a workflow which allows for the identification of large numbers of phosphorylation sites with minimal effort. In order to minimize experimental


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variation induced by sample preparation we prepared ~70 mg of HeLa whole cell lysate digest and stored it after desalting as dried aliquots of 3 mg each at -20°C. We first set a benchmark following the well-established protocol by Villen et al.25 Instead of IMAC we used TiO2 phosphopeptide enrichment as it performed better in our hands, resulting in higher numbers of identified phosphorylation sites and less unspecific enrichment of unphosphorylated peptides (data not shown). Using SCX chromatography, we generated 12 fractions and enriched each of them using TiO2 (SCX-TiO2) followed by LC-MSMS analysis. With this approach, we were able to identify 10908 non-redundant phosphorylation sites with a purity of 93% (Figure S1, Table S1). The most laborious step of this approach is the lyophilization, desalting, and phosphopeptide enrichment of the fractions generated during SCX fractionation, as every step has to be carried out 12 times and fractions are of high volume and complexity. We therefore tested if it is possible to perform first phosphopeptide enrichment and then fractionation by SCX chromatography. This would allow to only conduct one phosphopeptide enrichment and to reduce the complexity of the obtained SCX fractions. We performed TiO2 phosphopeptide enrichment of 3 mg whole cell lysate tryptic digest and fractionated the phosphopeptide enriched sample using SCX (TiO2-SCX) followed by LC-MSMS analyses using the same setup as used for the SCX-TiO2 experiments. We identified just 1291 phosphorylation sites showing a dramatically decreased performance (Figure S1, Table S1). This may be due to loss of phosphopeptides in the chromatographic system caused by unspecific interaction with metal ions present on surfaces of the LC-system and in the chromatographic column6 or decreased enrichment efficiency due to the high complexity of the sample. To investigate if the enrichment of phosphopeptides from the unfractionated samples led to lower enrichment efficiencies, and


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therefore lower numbers of identifications, we performed 4 hours gradient LC-MSMS analyses of unfractionated phosphopeptide enriched samples. Performance of single gradient analysis of enriched phosphopeptides is limited by the binding capacity of the analytical C18 column The binding capacity of the analytical C18 columns in our LC-MSMS setup should be in the range of several µg of peptide. We, therefore, questioned if 3 mg of peptide is too much as starting material for phosphopeptide enrichment when analyzing the unfractionated sample by 4 hr gradients, as the amount of enriched phosphopeptides may exceed the capacity of the column. We, therefore, performed phosphopeptide enrichment of 0.5, 1, 2, and 3 mg whole cell lysate digest using TiO2 and analyzed the resulting phosphopeptide-enriched fractions by LC-MSMS. We were able to identify 1349, 3980, 4984 and 5061 phosphorylation sites for 0.5, 1, 2, and 3 mg of cell lysate digest respectively (Figure S2). We had the impression, that the capacity of the system was already approaching its maximum using phosphopeptides enriched from 1 mg of starting material as doubling and tripling this amount both resulted only in an increase of identified phosphorylation sites by ~25% with virtually no difference between 2 mg and 3 mg of starting material. We achieved for 3 of the 4 samples a purity of more than 80% observing a slight reduction with increasing amounts of starting material: 86.2%, 84.6%, 81.3%, and 77.1% phosphopeptides for 0.5 mg, 1 mg, 2 mg, and 3 mg, respectively. Compared to the SCX-TiO2 sample we achieved similar enrichment efficiencies. We, therefore, conclude that the reduced number of peptides identified when performing TiO2-SCX was rather due to losses in the chromatography system than a decreased performance of TiO2 enrichment. Furthermore, analysis of batch-enriched samples with long LC gradients does not seem to be an appropriate alternative for our LC-MS system as we were not able to identify more than ~50% of the phosphorylation


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sites identified with the SCX-TiO2 strategy. We therefore evaluated tip-based fractionation methods, as it is possible to miniaturize the chromatography conditions by this approach, which should result in virtually no loss of phosphopeptides and allows for using large amounts of starting material. Fractionation of batch-enriched phosphopeptides using tip-based columns We evaluated three different types of chromatographic material covering the most common resins used for the preparation of tip-based chromatography columns: strong anion exchange (SAX)16, strong cation exchange (SCX)27, and C18 reversed phase material18 for basic reversed phase (BRP) fractionation (Figure 1).

Figure 1.Schematic view of the workflow for phosphopeptide enrichment and tip-based fractionation methods. HeLa whole cell lysates were digested by trypsin, 3mg of digest were enriched for phosphopeptides by TiO2 followed by a second round of TiO2 enrichment for the elution fraction of the first enrichment. Posphopeptide-enriched fractions were then subjected to fractionation using tip columns with either SAX, SCX or BRP material. SAX: strong anion exchanger; SCX: strong cation exchanger; BRP: basic reversed phase


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We generated tip-columns following the principle of STAGE tips22 using an increased number of disks in order to allow for higher binding capacity. SAX and BRP tip fractionation was carried out following published protocols16,26 employing decreasing pH value (SAX) and increasing acetonitrile content (BRP) for elution of peptides, respectively. For SCX tips we designed our own buffers reflecting the gradient we used for SCX fractionation of whole cell lysate digests with the chromatography system (SCX-TiO2), eluting peptides with increasing salt concentration. For each approach, we fractionated the enriched phosphopeptides from 3 mg whole cell lysate tryptic digest to 6 fractions. Additionally, we performed fractionation using a SCX column in combination with a chromatography system generating 6 fractions which were then enriched for phosphopeptides in order to be able to compare the performance of our approach to the common SCX-TiO2 procedure (column-SCX, CSCX). Each experiment was carried out in 3 independent replicates and eluate fractions were analyzed by LC-MSMS. SCX tip based fractionation outperforms the other approaches SCX tips resulted with 9812 identified phosphorylation sites on average in the highest number of identifications followed by CSCX, BRP tips and SAX tips with 9354, 5740 and 3950 sites, respectively (Figure 2A, Table S1). This correlates with the identification of 3674, 3868, 3611, and 2392 phosphoproteins. Out of these 5315 phosphoproteins in total, 34,2% overlap with a dataset published by Olsen et al.32 which performed SCX-TiO2 based analysis of the HeLa phosphoproteome while the phosphoproteins identified in the individual approaches overlap between 26% and 35% with these data (Figure S3).


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Figure 2. (A) Identified phosphorylation sites for SCX, SAX and BRP tip-fractionated batch-enriched phosphopeptides. CSCX samples were first fractionated followed by phosphopeptide enrichment of the single fractions. (B) Identified unique multiply phosphorylated peptides for each of the tip-based methods as well as CSCX. Shown are mean values + SD; n=3. SAX: strong anion exchanger; SCX: strong cation exchanger; BRP: basic reversed phase; CSCX: column chromatography SCX

For both SCX and BRP we identified 54% of phosphopeptides in all 3 replicates. The CSCX and SAX fractionated samples resulted in slightly lower values identifying 51% and 45% of phosphopeptides in all replicates, respectively (Figure S4A). When comparing the overlap between the different approaches, out of ~16,400 phosphosites identified in total, only 17% were detected with all 4 approaches. CSCX and SCX tips led to the identification of the highest number of unique phosphopeptides (17.7% and 17.3%) followed by BRP (6.1%) and SAX (5.3%), respectively (Figure S4B). For multiply phosphorylated peptides, SCX, and CSCX performed similar. They outperformed the other two methods by leading on average to 3.6


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(SAX) and 1.8 (BRP) times the number of identified multiply phosphorylated peptides (Figure 2B). The distribution of identified phosphopeptides across fractions (Figure 3A) followed for SAX and BRP a similar pattern with the first fraction resulting in the highest number of identifications (~25% of identified phosphopeptides), while all following fractions resulted in substantially lower numbers. The only exception was the last BRP fraction which also contained a high number of phosphopeptides. This is most likely due to the fact that it was composed of the first (flow-through) and the last fraction of elution (see Experimental section), indicating that a substantial amount of phosphopeptides does not bind to the C18 material under these conditions, as the last fraction (elution with 100% ACN) should not contain a high amount of phosphopeptides.

Figure 3. (A) Distribution of phosphorylated peptides across six fractions of the SAX, SCX and BRP tipbased fractionated batch enriched phosphopeptides as well as CSCX fractionated samples followed by enrichment of the individual fractions (displayed as percentage of the total number of identified phosphopeptides). (B) Distribution of multiply phosphorylated peptides across the six fractions of each method. Shown are mean values + SD; n=3. SAX: strong anion exchanger; SCX: strong cation exchanger; BRP: basic reversed phase; CSCX: column chromatography SCX


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For the SCX samples, the distribution was more even with the second fraction containing the highest number of phosphopeptides. CSCX fractionated samples showed a high amount of phosphorylated peptides in the first 3 fractions while identifications in the following 3 fractions decreased substantially. These results were quite surprising to us, as we expected the SAX material to bind the acidic phosphorylated peptides very efficiently33 and therefore did not expect to find the highest numbers of phosphopeptides in the flow-through. It seems that the phosphate group is not able to result in strong binding to the ion exchanger resin for all peptides. This may be improved by changing the buffer system used. For multiply phosphorylated peptides (Figure 3B) we observed a similar behavior as for singly phosphorylated peptides for the BRP samples, while in this case the presence of phosphopeptides in the fraction consisting of the flow through and the last elution was even more pronounced. In the SCX samples, the majority of multiply phosphorylated peptides eluted in the first 2 fractions, which is in accordance with the literature.34 In the CSCX fractionated samples the initial fraction contained the highest number of peptides, which was decreasing steadily with increasing fraction number. SAX, surprisingly, performed worst and no big differences for the numbers of identified multiply phosphorylated peptides between the individual fractions were observed. A possible reason for this bad performance is a very strong binding of multiply phosphorylated peptides to the SAX resin preventing their elution with the buffers used in our experimental setup. In general, the large differences observed between the different resins were quite surprising to us as the input and the number of fractions was the same for all approaches. Especially the results obtained for the BRP fractionated samples were quite unexpected as it has been shown before that BRP outperforms SCX in the classical “fractionation followed by enrichment” format.35,36 A possible reason for these discrepancies may be the material used for generating the BRP tip columns as different


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resins may exhibit varying stabilities under the basic conditions used. Furthermore, we only generated 6 fractions with discontinuous steps increasing by 2% ACN each for elution of phosphopeptides while most column-based approaches employ continuous gradients and collect higher numbers of fractions. This also results in a substantially different concatenation pattern compared to our approach possibly improving separation power and reducing redundancy. For the strong differences observed between SAX and SCX/CSCX fractionated samples, the presence of salt in the elution buffer may be essential and the use of salt gradients for SAX tips may therefore improve their performance. In order to further evaluate possible reasons for the discrepancies observed between the SAX, SCX, BRP, and CSCX fractionated samples, we performed gene ontology (GO) analyses of the phosphoproteins identified in the individual data sets (Figure S5, Table S3). We could not observe any differences in the GO categories assigned. This indicates that the physicochemical properties of the proteins from which the identified peptides originate are not likely to be a reason for the discrepancies observed. We therefore argued that the observed effects must be due to the properties of the individual peptides and their interaction with the resins in the different approaches. Identification Efficiency of Fragment Ion Spectra A possible reason for the observed discrepancies is differences in data acquisition. We therefore investigated the number of MSMS spectra acquired which was lowest for SCX and highest for CSCX (Figure 4A). In terms of identified MSMS spectra, however, the SCX fractionated samples outperformed the other samples resulting in an increase of 15%, 18% and 45% compared to BRP, CSCX, and SAX, respectively (Figure 4B).


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Figure 4. (A) Acquired MSMS spectra for SAX, SCX and BRP tip fractionated batch enriched phosphopeptides as well as CSCX fractionated samples followed by enrichment of the individual fractions. For each approach, 3 mg of tryptic HeLa whole cell lysate digest was used. (B) Identified MSMS spectra for each fractionation method. Shown are mean values + SD; n=3. SAX: strong anion exchanger; SCX: strong cation exchanger; BRP: basic reversed phase; CSCX: column chromatography SCX

A possible reason for these observations are differences in signal intensity for the fragmented phosphopeptides as high-intensity precursor ions will lead more likely to the identification of the respective peptides. Possible reasons leading to higher intensities are i) a better distribution of high abundant ions across fractions in the SCX samples resulting in lower complexities of individual fractions and therefore no under sampling in the data dependent acquisition (DDA) mode, or ii) a better resolution leading to higher concentrations of peptides in single fractions


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and preventing redundant fragmentation. For the SAX, BRP, and CSCX fractionated samples we observed a decrease of acquired MSMS spectra with increasing fraction number while no such effect could be seen for SCX (Figure S6) indicating a more homogeneous distribution of phosphopeptides for the SCX fractionated samples. Additionally, we observed higher redundancy in the SAX fractionated samples compared to SCX and BRP which resulted in roughly similar values (Figure S7): more than 50% of the peptides in the SAX fractionated samples were detected more than 4 times. This implies that SAX fractionation leads to a broader distribution of peptides across fractions. This results in reduced signal intensities in the individual fractions and therefore reduces the likelihood for fragmentation (and identification) in the LC-MSMS experiments. For CSCX fractionated samples, however, the redundancy was significantly lower across all datasets showing superior fractionation compared to all tip based approaches. It also, however, indicates a higher loss of phosphorylated peptides during sample preparation as the reduced redundancy should facilitate the identification of more phosphorylation sites compared to the tip based SCX approach, which is not the case. in the tip based approaches, a broader dilution of the sample should lead to more features identified, since the same peptide will be recognized in the survey scan of multiple runs. We therefore investigated how many peptide features were detected by MaxQuant in the SAX, SCX, BRP, and CSCX fractionated samples (Figure S8). The SAX fractionated samples resulted in ~22000 and the BRP fractionated samples in ~31000 features more than the SCX and CSCX fractionated samples which resulted in similar values (increase of 9%and 12% relative to SCX and CSCX, respectively) confirming a broader distribution of peptides for the SAX and BRP tip fractionated samples. Identification of non-phosphorylated peptides


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Next, we investigated the purity of phosphopeptides by comparing numbers of identified phosphorylated and non-phosphorylated peptides. Surprisingly, the purity varied strongly between SAX (62%), SCX (86%), BRP (66%) and CSCX (84%) fractionated samples (Figure 5).

Figure 5. (A) Average number of phosphorylated and non-phosphorylated peptides identified in SAX, SCX and BRP tip fractionated samples of batch enriched phosphopeptides as well as CSCX fractionated samples in combination with phosphopeptide enrichment of the single fractions. For each experiment, 3 mg of HeLa whole cell tryptic digest was used. (B) Purity of phosphopeptide enriched fractions (%) for the different fractionation methods. Shown are mean values + SD; n=3. SAX: strong anion exchanger; SCX: strong cation exchanger; BRP: basic reversed phase; CSCX: column chromatography SCX


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Across single fractions, the percentage of phosphorylated peptides was constantly ~60% for SAX fractionated samples but varied between fractions of the SCX (97% to 80%),BRP (84% to 43%), and CSCX (96% to 46%) fractionated samples (Figure S9). The strong difference between fractions we observed in the BRP samples reflects the influence of phosphate groups on retention behavior in reversed phase chromatography. Peptides carrying a phosphate group elute earlier due to their increased hydrophilicity allowing unspecifically enriched non-phoshorylated peptides to be detected in the later fractions. Earlier elution of phosphorylated peptides compared to their unmodified counterparts can also be seen for C18 reversed phase material under acidic conditions.34 The discrepancies between SCX and CSCX were quite surprising to us since we used the same solutions for elution of peptides in both approaches. They are most likely due to a stronger binding of the stationary phase used in the tip based approaches. The higher redundancy of SCX compared to CSCX cannot explain the observation, as just the number of phosphopeptides identified solely in fraction 6 of the SCX samples was higher than all combined identifications in the last fraction of the CSCX experiment. Analysis of Fraction-specific Peptide Properties In order to better understand the broad distribution of phosphopeptides across SAX fractions and the high variations in phosphopeptide purity, we investigated the isoelectric point, the length and the number of missed cleavage sites of the identified peptides across the fractions of the different approaches. Especially for SAX, we observed a strong correlation of the fraction number and the isoelectric point (Figure S10) of the peptides while acidic peptides presented the most abundant population. The BRP fractionated samples contained more neutral and basic peptides while the SCX and CSCX samples resulted in a rather uniform distribution. When investigating the missed cleavage sites, we observed roughly similar cleavage rates for the SAX and BRP fractionated


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samples (Figure S11). For SAX, the rate of miscleaved peptides increased with the fraction number while the opposite was the case for the BRP samples. The SCX samples showed significantly lower miscleavage rates and a clear distribution with small amounts of miscleaved peptides in the first fraction (15% and 22%) which increased steadily to high numbers in the last fraction (64% and 55%) for the phosphorylated and the non-phosphorylated peptides, respectively. This is most likely due to the increased interaction of the SCX material with the additional basic amino acid which is present in the miscleaved peptides. A similar trend was observed for the CSCX samples with a higher number of observed missed cleavage sites both for phosphorylated (20% to 77%) and non-phosphorylated peptides (36% to 79%). In total, as expected, the percentage of missed cleavage sites among phosphopeptides was higher than among non-phosphopeptides.37 When investigating the length of peptides (Figure S12), we found that SCX and CSCX identified on average shorter peptides than BRP and SAX. A possible reason may be that the interaction with the stationary phase in SAX and BRP is due to the whole peptide backbone while it is in SCX mainly based on the basic groups present at the N- and Cterminus of tryptic peptides. Differences in identification efficiency are due to retention of short peptides and differences in resolution Based on the results of our analyses, we have the impression that for the tip format, the SCX material is superior to SAX and BRP due to a more efficient distribution of the phosphorylated peptides across the single fractions. This results in higher intensities and lower fraction complexity and therefore a better identification in the DDA mode. We believe that the underrepresentation of short phosphopeptides in SAX and BRP is due to an insufficient interaction with the chromatographic material resulting in highly complex initial fractions and


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therefore undersampling in these LC-MSMS runs. Longer peptides are retained more efficiently but are also not fractionated with a sufficient resolution resulting in elution across several fractions. This results in redundant identification of abundant peptides and dilution effects reducing precursor intensities of low abundant ions preventing their identification. This favors the identification of unphosphorylated background peptides leading to the observed reduced phosphopeptide purity in SAX and BRP, which is less pronounced in the SCX samples. CSCX fractionated samples show similar characteristics and performance compared to such fractionated by SCX tips. The main difference is the reduced redundancy of CSCX which, however, fails to result in increased numbers of identified phosphorlylation sites. We believe this is due to an increased loss of phosphopeptides during sample fractionation and enrichment in CSCX and a better retention of phosphopeptides on the tip SCX resin.

Conclusions We show that the fractionation of phosphopeptides using pipet tip-based columns leads to similar results as the common approach using liquid chromatography based approaches at a fraction of costs and time. It is not possible to completely omit a fractionation step since single run analyses approach a maximal number of identifiable peptides which is most likely due to a limited capacity of the analytical C18 column. Furthermore, fractionation of batch-enriched phosphopeptides using a chromatography system was in our hands not practical, which is most likely due to unspecific losses of phosphopeptides during fractionation. This may be less pronounced if a dedicated chromatography system is present for these analyses as it was shown before that such a setup can work efficiently.15,17,18 For tip-based fractionation, SCX outperforms


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SAX and BRP clearly, leading to an increase in the number of identified phosphorylation sites of 148% and 71%, respectively. We believe that this is due to a stronger interaction of the phosphopeptides with the SCX material leading to a more efficient retention and a better resolution. Since the individual resins cover different fractions of the phosphoproteome, the number of identified phosphorylation sites of a sample can be expanded by fractionating it with several tip-based columns. Especially for research groups which are not specialized in the analysis of phosphopeptides, the tip based fractionation of batch-enriched phosphopeptides should be of value as it allows performing large-scale phosphoproteomics experiments even for unexperienced operators. Compared to the common approach, which requires expensive chromatography systems and SCX columns as well as high amounts of desalting cartridges, plastic ware, and TiO2, the tip-based system comes at a fraction of costs and offers high robustness, simplicity and reproducibility. Furthermore, the sample processing time is reduced from one week to only 2 days at a comparable performance. Since all materials involved are single use items, there is no possibility for carry over increasing the purity of samples processed together and it is possible to fractionate high amounts of samples in parallel. We therefore believe, that the fractionation of batch-enriched phosphopeptides using SCX tip-based columns is a valuable approach for the large-scale characterization of phosphopeptides from complex samples.

SUPPORTING INFORMATION: The following files are available free of charge at ACS website http://pubs.acs.org: Figure S1: Comparison between SCX-HPLC-TiO2 and TiO2-SCX-HPLC for enrichment and fractionation.


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Figure S2: Phosphorylation sites identified in the analysis of unfractionated samples. Figure S3: Comparison of published data to the phosphoproteins identified using the different fractionation methods. Figure S4: Reproducibility of different tip-based methods and the Column SCX approach. Figure S5: Gene ontology analysis of phosphoproteins identified using the different fractionation methods. Figure S6: Number of acquired MSMS spectra for the different fractionation methods. Figure S7: Comparison of peptide redundancy in the different fractionation methods. Figure S8: Number of peptide features detected in different fractionation methods. Figure S9: Comparison of phosphopeptide purity for the different fractionation methods. Figure S10: Comparison of the theoretical isoelectric points for peptides detected in the different fractionation methods. Figure S11: Distribution of miscleaved peptides across individual fractions of the different fractionation methods. Figure S12: Comparison of the peptide length across individual fractions of the different fractionation methods. Table S1: Phosphorylation sites acquired from all experiments and all biological replicates. Table S2: Phosphopeptides identified in each individual experiment from the SAX, SCX, and BRP tip-based methods and the column chromatography fractionation experiments (CSCX). Table S3: Gene ontology results for the individual datasets obtained from PANTHER.

AUTHOR INFORMATION Corresponding Author


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* Dominic Winter, email: [email protected]; Phone: +49 228 737081; Fax: +49 228 732416 Orcid Dominic Winter: 0000-0001-6788-6641. AlirezaDehghani: 0000-0003-1577-1038 Author Contributions AD+MG performed experiments, DW+AD designed experiments, AD+DW wrote the manuscripts Notes The Authors declare no competing financial interest. ACKNOWLEDGMENTS We would like to thank the German Academic Exchange Service (DAAD) and the Marie Curie Programme of the European Union for Financial Support.

ABBREVIATIONS SCX, strong cation exchange; SAX, strong anion exchange; BRP, basic reversed phase, TiO2titanium dioxide; ACN, acetonitrile; AcOH, acetic acid; TFA, trifluoroacetic acid; FA, formic acid; LC-MS, liquid chromatography–mass spectrometry


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Table of Contents Graphic (TOC):


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Tip based fractionation of batch-enriched phosphopeptides facilitates

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