# Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis

Núria Colomé, Joaquín Abian, Kerman Aloria, Jesús Arizmendi, Silvia Barceló-Batllori, Sophie Braga-Lagache, Odile Burlet-Schiltz, Montse Carrascal, J Ignacio Casal, Eduard Chicano-Gálvez, et al.

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Núria Colomé, Joaquín Abian, Kerman Aloria, Jesús Arizmendi, Silvia Barceló-Batllori, et al. Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis. Journal of Proteomics, 2022, 251, pp.104409. 10.1016/j.jprot.2021.104409. hal-04998186

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Multi-laboratory experiment PME11 for the standardization of
phosphoproteome analysis

Núria Colomé<sup>a</sup>, Joaquín Abian<sup>b</sup>, Kerman Aloria<sup>c</sup>, Jesús M. Arizmendi<sup>d</sup>, Silvia Barceló-Batllori<sup>e</sup>, Sophie Braga-Lagache<sup>f</sup>, Odile Burlet-Schiltz<sup>g</sup>, Montse Carrascal<sup>b</sup>, J. Ignacio Casal<sup>h</sup>, Eduard Chicano-Gálvez<sup>i</sup>, Cristina Chiva<sup>j,k</sup>, Luis Felipe Clemente<sup>l</sup>, Felix Elortza<sup>m</sup>, Josep M. Estanyol<sup>n</sup>, Joaquín Fernandez-Irigoyen<sup>o</sup>, Patricia Fernández-Puente<sup>p</sup>, María José Fidalgo<sup>n</sup>, Carine Froment<sup>g</sup>, Manuel Fuentes<sup>q</sup>, Carlos Fuentes-Almagro<sup>r</sup>, Marina Gay<sup>s</sup>, Alexandre Hainard<sup>t</sup>, Manfred Heller<sup>f</sup>, María Luisa Hernández<sup>l</sup>, Nieves Ibarrola<sup>u</sup>, Ibon Iloro<sup>m</sup>, Thomas Kieselbach<sup>v,1</sup>, Antonio Lario<sup>w</sup>, Marie Locard-Paulet<sup>g</sup>, Anabel Marina-Ramírez<sup>x</sup>, Luna Martín<sup>a</sup>, Esperanza Morato-López<sup>x</sup>, Javier Muñoz<sup>y</sup>, Rosana Navajas<sup>z</sup>, M. Antonia Odena<sup>aa</sup>, Leticia Odriozola<sup>ab</sup>, Eliandre de Oliveira<sup>aa</sup>, Alberto Paradela<sup>z</sup>, Carla Pasquarello<sup>t</sup>, Vivian de los Rios<sup>h</sup>, Cristina Ruiz-Romero<sup>ac</sup>, Eduard Sabidó<sup>j,k</sup>, Manuel Sánchez del Pino<sup>ad</sup>, Jaime Sancho<sup>w</sup>, Enrique Santamaría<sup>o</sup>, Christine Schaeffer-Reiss<sup>ae</sup>, Justine Schneider<sup>ae,2</sup>, Carolina de la Torre<sup>e</sup>, M. Luz Valero<sup>af</sup>, Marta Vilaseca<sup>s</sup>, Shuai Wu<sup>ag</sup>, Linfeng Wu<sup>ag</sup>, Pilar Ximénez de Embún<sup>y</sup>, Francesc Canals<sup>a,**</sup>, Fernando J. Corrales<sup>z,ab,*</sup>, ProteoRed-ISCIIIz'ah, EuPAz'ai

<footnote><sup>a</sup> ProteoRed-ISCIII, Vall d'Hebron Institute of Oncology (VHIO), Barcelona 08035, Spain</footnote>

<footnote><sup>b</sup> ProteoRed-ISCIII, Instituto de Investigaciones Biomédicas de Barcelona, IBB-CSIC/IDIBAPS, 08036 Barcelona, Spain</footnote>

<footnote><sup>c</sup> ProteoRed-ISCIII, Proteomics Core Facility-SGIKER, University of the Basque Country (UPV/EHU), Leioa, Spain</footnote>

<footnote><sup>d</sup> Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain</footnote>

<footnote><sup>e</sup> ProteoRed-ISCIII, Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain</footnote>

<footnote><sup>f</sup> Department for BioMedical Research (DBMR), Proteomics and Mass Spectrometry Core Facility, University of Bern, CH-3010 Bern, Switzerland</footnote>

<footnote><sup>g</sup> Proteomics and Mass Spectrometry of Biomolecules, Proteomics Infrastructure of Toulouse, Proteomics French Infrastructure, ProFI. Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, UPS, CNRS, Toulouse, France</footnote>

<footnote><sup>h</sup> ProteoRed-ISCIII, Centro de Investigaciones Biológicas-CSIC, Madrid 28040, Spain</footnote>

<footnote><sup>i</sup> ProteoRed-ISCIII, Proteomics Unit, IMIBIC/UCO/HURS, IMIBIC Building Fl.3, 14004 Córdoba, Spain</footnote>

<footnote><sup>j</sup> Proteomics Unit, Center for Genomics Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona, Spain</footnote>

<footnote><sup>k</sup> ProteoRed ISCIII, Proteomics Unit, Universitat Pompeu Fabra, Barcelona, Spain</footnote>

<footnote><sup>l</sup> ProteoRed-ISCIII, Proteomics Unit, Complutense University, 28040 Madrid, Spain</footnote>

<footnote><sup>m</sup> ProteoRed-ISCIII, CIC bioGUNE, Proteomics Platform, Basque Research & Technology Alliance (BRTA), CIBERehd,Bizkaia Science and Technology Park, 48160 Derio, Spain</footnote>

<footnote><sup>n</sup> ProteoRed-ISCIII, Scientific and Technological Centers (CCITUB), University of Barcelona, 08036 Barcelona, Spain</footnote>

<footnote><sup>o</sup> ProteoRed-ISCIII, Proteomics Unit, Clinical Neuroproteomics Group, Navarrabiomed, Complejo Hospitalario de Navarra (CHN), Universidad Pública de Navarra (UPNA), IdiSNA, 31008 Pamplona, Spain</footnote>

<footnote><sup>p</sup> Grupo de Investigación de Reumatología (GIR), Agrupación CICA-INIBIC, Universidad de A Coruña, A Coruña, Spain</footnote>

<footnote><sup>q</sup> Department of Medicine and General Cytometry Service-Nucleus, Proteomics Unit, CIBERONC, Cancer Research Center (IBMCC/CSIC/USAL/IBSAL), Universidad de Salamanca, Spain</footnote>

<footnote><sup>r</sup> Proteomics Unit, SCAI, University of Córdoba, Ramón y Cajal Building, Rabanales Campus, 14071, Córdoba, Spain</footnote>

<footnote><sup>s</sup> ProteoRed-ISCIII, Institute for Research in Biomedicine (IRB Barcelona), BIST (The Barcelona Institute of Science and Technology), Baldiri i Reixac 10, 08028 Barcelona, Spain</footnote>

<footnote><sup>t</sup> Proteomics Core Facility, CMU, University of Geneva, Switzerland</footnote>

<footnote><sup>u</sup> ProteoRed-ISCIII, Proteomics Unit, Cancer Research Center (IBMCC/CSIC/USAL/IBSAL), Universidad de Salamanca-CSIC, Salamanca, Spain</footnote>

<footnote><sup>v</sup> Umeå University, Department of Chemistry, 90187, Umeå, Sweden</footnote>

<footnote><sup>w</sup> ProteoRed-ISCIII, IPBLN-CSIC, 18016 Granada, Spain</footnote>

<footnote>* Corresponding author at: ProteoRed-ISCIII, Centro Nacional de Biotecnologia (CSIC), 28049, Madrid, Spain.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: fcanals@vhio.net (F. Canals), fcorrales@cnb.csic.es (F.J. Corrales).</footnote>
x ProteoRed-ISCIII, CBM Severo Ochoa (CSIC-UAM), Madrid 28049, Spain

y ProteoRed-ISCIII, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain

z ProteoRed-ISCIII, Centro Nacional de Biotecnología (CSIC), 28049, Madrid, Spain

aa ProteoRed-ISCIII, Proteomics Platform, Barcelona Science Park, 08028, Barcelona, Spain

ab ProteoRed-ISCIII, CIMA, University of Navarra, 31008, Pamplona, Spain

ac Grupo de Investigación de Reumatología (GIR) - ProteoRed-ISCIII, Unidad de Proteómica, INIBIC-Complejo Hospitalario Universitario de A Coruña, SERGAS, A Coruña, Spain

ad Biotechnology and Biomedicine Interdisciplinary Research Unit (ERI BIOTECMED), University of Valencia, 46100 Burjassot, Spain

ae Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000, Strasbourg, France

af ProteoRed-ISCIII, Proteomics Unit, Central Service for Experimental Research (SCSIE), University of Valencia, 46100, Burjassot, Spain

ag Agilent Technologies, Inc., Santa Clara, CA 95051, USA

ah ProteoRed-ISCIII-PRB3, Spanish Proteomics Networked Platform, Centro Nacional de Biotecnología (CSIC), 28049, Madrid, Spain

ai European Proteomics Association, Standardization Initiative, Centro Nacional de Biotecnología (CSIC), 28049, Madrid, Spain

## ABSTRACT

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

Many aspects of cell biology are regulated by reversible protein phosphorylation networks that involve thousands of phosphorylation events. In the last decade multiple methods have been developed to identify and quantify involved phosphorylation sites, and their modulation and dynamics under physiological and pathological conditions. Global post-translational modification analysis based on cutting edge mass spectrometry technology has emerged as the premier tool in many laboratories worldwide to investigate the complexity of signaling pathways and their crosstalk [1-3].

In 2016 the Spanish Proteomics Network ProteoRed-ISCIII proposed the PME11 multi-laboratory experiment as part of the EuPA Standardization Initiative. The aim was to evaluate the performance and reproducibility of phosphopeptide enrichment procedures and to test the usefulness of phosphopeptide mixture standards to set up, monitor, and troubleshoot phosphopeptide analysis pipelines. The reference samples analyzed in the study (PME11-A1, A2, A3) consisted of a yeast tryptic digest (125 µg of a C-18 purified peptide digest), spiked-in with three different concentrations (100, 250 and 500 fmol) of a mixture of 20 human phosphopeptide standards (Phosphomix 1 and 2 from Sigma-Aldrich, (product reference MSPL1 and MSPL2, Table 1), containing light isotopes. Each participant laboratory received two aliquots of each of the three samples (SUPP INFO 1&2), that were distributed in dry ice, lyophilized from a water-acetonitrile mixture. One additional vial PME11-B, containing 2 pmol of each of the corresponding isotopically labeled heavy Phosphomix standard peptides (Sigma-Aldrich MSP1H and MSP2H) was distributed in dried form for ulterior quantitative analyses. Upon reception participants were indicated to re-dissolve the samples in the appropriate buffer for the enrichment procedure selected. Then, enriched phosphopeptides were analyzed by LC-MS/MS (three replicates) following the recommended guidelines (10 to 30% of the enriched sample and 60 min 0-35% acetonitrile gradient). Analysis of pre-enriched samples was also recommended. Detailed descriptions of the experimental settings, reference sample and analysis guidelines were provided to the participants (SUPP INFO 1&2).

Recently, a related study conducted by several laboratories in the frame of the MS Resource Pillar of the HUPO Human Proteome Project has been reported [4]. In this study, a standard set of 94 phosphopeptides and their nonphosphorylated counterparts, mixed in a neat sample and a yeast background were analyzed. Unlike the HUPO study samples, the samples proposed in the present study allowed for the assessment of the enrichment of the endogenous yeast phosphopeptides, in conditions and amounts similar to a real sample. Besides, the spiked-in phosphopeptide standards were provided in isotopically labeled and unlabeled form, allowing for assessment not only of targeted phosphopeptide analysis, but also to estimate the yield of the enrichment procedures used.

Under the coordination of ProteoRed-ISCIII, 36 datasets were received from 23 laboratories (Table 2) distributed across Europe -Spain, France, Switzerland, United Kingdom, and Sweden- and USA. Individual reports including experimental details and results were prepared by each participant in the template specifically designed for this experiment. Additionally, MS/MS files (mgf format) were also submitted to the coordination unit for their centralized processing and integration, which will be described elsewhere. Some laboratories provided various datasets that corresponded to different analytical pipelines, which allowed the specific evaluation of the experimental conditions tested as the user and instrument used in these cases were the same. Shotgun analysis results were used to evaluate the general performance of each laboratory in terms of number of yeast phosphopeptides identified, efficiency of the enrichment procedure (phosphopeptides/total peptides ratio) and detection of spiked-in phosphopeptide standards.

In light of the dispersion of the analytical conditions used by the participating labs, a comprehensive statistical analysis may have limitations. Nevertheless, several outcomes are worth to be discussed taking into consideration the interlaboratory nature of the present experiment. Samples were processed following different protocols in eight different mass spectrometers as summarized in Table 2 and supplementary information.

A first clear outcome is that intra-laboratory reproducibility is in general very good, as shown by the error bars in the graph in Fig. 1A, with a median %CV between triplicate analysis of 9.16% (Table 2). It has to be remarked that these correspond to triplicate experiments, including the enrichment step and the LCMS analysis.

Regarding inter-laboratory comparison, the number of

<footnote n="1">¹ Current affiliation: Umeå University Library, Scientific Communication, 901 74 Umeå, Sweden</footnote>

<footnote n="2">² Current affiliation: Inoviem Scientific, Bioparc3, 850 Boulevard Sébastien Brant, 67400, Illkirch-Graffenstaden, France</footnote>
<table><caption>**Table 1**
Phosphopeptide standard description.</caption><thead><tr><th>Sequence</th><th>PhosphoMix #</th><th>phosphosite 1</th><th>phosphosite 2</th></tr></thead><tbody><tr><td>ADEPSSEESDLEIDK</td><td>1_6</td><td>S5</td><td></td></tr><tr><td>ADEPSSEESDLEIDK</td><td>1_7</td><td>S6</td><td>S9</td></tr><tr><td>ADEPSSEESDLEIDK</td><td>2_6</td><td>S9</td><td></td></tr><tr><td>ELSNSPRENSFGSPLEFR</td><td>1_9</td><td>S5</td><td>S14</td></tr><tr><td>ELSNSPRENSFGSPLEFR</td><td>2_9</td><td>S3</td><td>S5</td></tr><tr><td>ETQSPEQVK</td><td>2_3</td><td>T2</td><td></td></tr><tr><td>EVQAEQPSSSSPR</td><td>1_5</td><td>S10</td><td></td></tr><tr><td>FEDEGAGFEESSETGDYEEK</td><td>1_8</td><td>S12</td><td></td></tr><tr><td>HQYSDYDYHSSSEK</td><td>2_7</td><td>Y8</td><td>S12</td></tr><tr><td>LGPGRPLPTPTSECTSDVEPDTR</td><td>2_10</td><td>T12</td><td></td></tr><tr><td>LPQETAR</td><td>2_1</td><td>T5</td><td></td></tr><tr><td>NTPSQHSHSIQHSPER</td><td>2_8</td><td>S4</td><td>S9</td></tr><tr><td>RDSLGTYSSR</td><td>1_3</td><td>T6</td><td></td></tr><tr><td>RSYSRSR</td><td>1_2</td><td>Y3</td><td>S4</td></tr><tr><td>RYSSRSR</td><td>2_2</td><td>S3</td><td>S4</td></tr><tr><td>SPTEYHEPVYANPFYRPTTPQR</td><td>1_10</td><td>Y10</td><td>T19</td></tr><tr><td>SRPSSPELNNK</td><td>2_5</td><td>S1</td><td>S5</td></tr><tr><td>TKLITQLRDAK</td><td>1_4</td><td>T1</td><td>T5</td></tr><tr><td>VIEDNEYTAR</td><td>2_4</td><td>Y7</td><td></td></tr><tr><td>VLHSGSR</td><td>1_1</td><td>S6</td><td></td></tr></tbody></table>

phosphopeptides identified in the different experiments spans a wide range, with an average value of 1026, (Fig. 1A. B and Table 2). One of the main factors that explains this wide range is of course the technical capability of the different instruments used. To roughly estimate the contribution of this factor, normalized values have been calculated (black points in the graph) using as normalization factor the ratio between the reported number of total peptides in the analysis of the pre-enrichment sample for each experiment (Table 2), and the average values for all the experiments. Using this normalization to “compensate” for instrument performance, the inter-laboratory %CV for the number of phosphopeptides decreases from 66% to 36% (Fig. 1B).

Other factors accounting for this variability would certainly include the enrichment protocol used, as well as the parameters used for data processing and database searches, but also reflect the different expertise of the different laboratories. This is apparent when comparing the results from laboratories using the same type of enrichment and identical instrument (see for example L14 vs L09, L28 vs L15, or L13 vs L20, in Fig. 1A).

The amount of sample analyzed (Table 2) is also a factor that influences the result, as is well illustrated by data from L23 with around 500 phosphopeptides detected upon processing 10% sample in an Orbitrap XL (L23-1 and -4) and about 1000 and 2000 identifications when 2% and 20% sample were processed (L23-2 and -3, -5 and -6 respectively) in an Orbitrap Fusion Lumos respectively (Fig. 1A and Table 2).

The enrichment selectivity (Fig. 1C) spans from 15 to 90%. Overall, there is no clear correlation between the observed selectivity and the number of phosphopeptides identified in each of the experiments, influenced, as discussed, by many other factors.

TiO₂ was the preferred enrichment method, representing more than 80% of the analyses and resulted in higher enrichment selectivity (above 50%) compared to those obtained with IMAC (below 40%). This is also the case when comparing TiO₂ versus IMAC enrichment data from the same laboratory, such as data from L23 and L13. These results seem to be in agreement with previous data reporting increased selectivity of

TiO₂ compared to metal chromatography [5]. It has been also reported that IMAC enrichment would favor the identification of phosphorylated peptides [6]. In the results gathered in this study, no significant differences have been observed in this respect (data not shown). However, the small number of IMAC analysis, together with the variety of protocols and instruments used, precludes a general conclusion. Combination of two enrichment steps, either TiO₂-TiO₂ or TiO₂-IMAC, increased the enrichment efficiency notwithstanding the total number of phosphopeptides identified (comparing data obtained in the same instrument in different labs), as deduced from L25 data (Fig. 1A and B).

Data from TiO₂ and IMAC enrichment for L23 and L13, but also data from L28, where different sample/TiO₂ matrix ratios were assayed (see Table 2), exemplify a general trend where higher selectivity of the enrichment step would result in a significant increase in the number of phosphopeptides detected. This behavior would be consistent with a “masking” effect of the presence of a higher ratio of non-phosphorylated peptides with respect to phosphopeptides in the enriched sample.

The enrichment chromatography format did not have any systematic effect either in the number of phosphopeptides detected or in the enrichment capacity; the observed variations result from inter-operator variability.

Detection of phosphopeptide standards relied on an enrichment step, no matter the amount of standard spiked on the yeast extract (aprox. 100, 50 or 20 fmol on column). The frequency of detection defined as the proportion of laboratories detecting a given peptide in three samples, was above 60% for most phosphopeptides (12/20 labs), around 50% in five cases while three phosphopeptides were not detected in any lab, likely due to their small size and highly hydrophilic nature, preventing their retention in the C18 precolumn (Fig. 2). No significant differences were observed in terms of phosphomix standard detection in regard of the different instruments or enrichment methods used. The phosphomix peptide standards are in general readily observable, even at the lowest concentration assayed, with the exceptions described, and so can be useful for quantitative purposes to measure the yield of a particular
<caption>**Table 2**</caption>

Datasets gathered in the study, experimental settings, and summary of the main results. Datasets coded with the same L number correspond to experiments performed in the same laboratory using different enrichment or LCMS analysis conditions.

<table><thead><tr><th>CODE</th><th>Affinity<br>Enrichment<br>type</th><th>Support</th><th>Ratio TiO2/<br>sample w/w</th><th>MS<br>instrument</th><th>Enriched sample<br>amount loaded in<br>LCMS (%)</th><th># peptides Pre-<br>enriched sample</th><th># Phospho-<br>peptides<sup>1</sup></th><th>Std Dev. #<br>Phospho-<br>peptides<sup>1</sup></th><th>%CV #<br>Phospho-<br>peptides<sup>1</sup></th></tr></thead><tbody><tr><td>L06</td><td>TiO2</td><td>Stage Tip<br>SpinTip<br>column</td><td>6</td><td>5600 TTOF</td><td>30</td><td>5755</td><td>823</td><td>59.2</td><td>7.2</td></tr><tr><td>L12</td><td>TiO2</td><td>Stage Tip<br>SpinTip<br>column</td><td>N/A</td><td>5600 TTOF</td><td>25</td><td>5666</td><td>679</td><td>28.4</td><td>4.2</td></tr><tr><td>L07</td><td>TiO2</td><td>Stage Tip<br>SpinTip<br>column</td><td>6</td><td>5600 TTOF<br>Amazon<br>Speed ETD</td><td>25</td><td>11,008</td><td>741</td><td>199.3</td><td>26.9</td></tr><tr><td>L16</td><td>TiO2</td><td>Stage Tip<br>SpinTip<br>column</td><td>8</td><td>Orbitrap XL</td><td>30</td><td>1461</td><td>606</td><td>19.1</td><td>13.5</td></tr><tr><td>L21_1</td><td>TiO2</td><td>Stage Tip<br>SpinTip<br>column</td><td>6</td><td>Orbitrap XL</td><td>10</td><td></td><td></td><td>101.9</td><td>16.8</td></tr><tr><td>L23_1</td><td>TiO2</td><td>Stage Tip<br>column</td><td>6</td><td>Orbitrap XL</td><td>15</td><td></td><td></td><td>39.7</td><td>7.4</td></tr><tr><td>L14_1</td><td>TiO2</td><td>Stage Tip<br>Batch<br>SpinTip<br>column</td><td>6</td><td>OT Fusion</td><td>15</td><td>9073</td><td>856</td><td>112.8</td><td>13.2</td></tr><tr><td>L14_2</td><td>TiO2</td><td>Batch<br>SpinTip<br>column</td><td>24</td><td>OT Fusion</td><td>15</td><td>9024</td><td>716</td><td>150.9</td><td>21.1</td></tr><tr><td>L09</td><td>TiO2</td><td>Batch<br>SpinTip<br>column</td><td>N/A</td><td>OT Fusion<br>OT Fusion</td><td>20</td><td></td><td></td><td>24.5</td><td>24.0</td></tr><tr><td>L28_1</td><td>TiO2</td><td>Batch<br>SpinTip<br>column</td><td>0.3</td><td>Lumos<br>OT Fusion</td><td>17</td><td>23,851</td><td>2675</td><td></td><td></td></tr><tr><td>L23_3</td><td>TiO2</td><td>Batch<br>SpinTip<br>column</td><td>6</td><td>Lumos<br>OT Fusion</td><td>20</td><td>12,402</td><td>2486</td><td>83.8</td><td>3.4</td></tr><tr><td>L19</td><td>TiO2</td><td>Batch<br>SpinTip<br>column</td><td>N/A</td><td>Lumos<br>OT Fusion</td><td>20</td><td>16,524</td><td>2295</td><td>54.7</td><td>2.4</td></tr><tr><td>L28_2</td><td>TiO2</td><td>Batch<br>SpinTip<br>column</td><td>40</td><td>Lumos<br>OT Fusion</td><td>17</td><td>23,851</td><td>2184</td><td></td><td></td></tr><tr><td>L28_3</td><td>TiO2</td><td>Batch<br>SpinTip<br>column</td><td>6</td><td>Lumos<br>OT Fusion</td><td>17</td><td>23,851</td><td>2161</td><td></td><td></td></tr><tr><td>L10</td><td>TiO2</td><td>Stage Tip<br>SpinTip<br>column</td><td>6</td><td>Lumos<br>OT Fusion</td><td>20</td><td>17,286</td><td>2062</td><td>57.4</td><td>2.8</td></tr><tr><td>L23_2</td><td>TiO2</td><td>Stage Tip<br>SpinTip<br>column</td><td>6</td><td>Lumos<br>OT Fusion</td><td>2</td><td>10,181</td><td>1104</td><td>26.7</td><td>2.4</td></tr><tr><td>L15</td><td>TiO2</td><td>Stage Tip<br>column</td><td>6</td><td>Lumos<br>OT Fusion</td><td>10</td><td>5636</td><td>768</td><td>34.9</td><td>4.6</td></tr><tr><td>L02_2</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Stage Tip<br>column</td><td>5</td><td>OT Velos</td><td>25</td><td>5813</td><td>1333</td><td>144.7</td><td>10.9</td></tr><tr><td>L13_1</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Stage Tip<br>column</td><td>6</td><td>OT Velos</td><td>20</td><td>5299</td><td>1184</td><td>56.7</td><td>4.8</td></tr><tr><td>L05</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Stage Tip<br>column</td><td>24</td><td>OT Velos</td><td>30</td><td>6846</td><td>1051</td><td>136.6</td><td>13.0</td></tr><tr><td>L04</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Stage Tip<br>column</td><td>8</td><td>OT Velos</td><td>30</td><td></td><td></td><td>67.6</td><td>11.3</td></tr><tr><td>L03</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Stage Tip<br>column</td><td>6</td><td>OT Velos</td><td>20</td><td>4194</td><td>586</td><td>206.0</td><td>35.1</td></tr><tr><td>L02_1</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Magnetic Beads<br>column</td><td>2</td><td>OT Velos</td><td>25</td><td>5813</td><td>549</td><td>92.9</td><td>16.9</td></tr><tr><td>L20</td><td>TiO2</td><td>Magnetic Beads<br>Stage Tip<br>Batch<br>Stage Tip<br>column</td><td>N/A</td><td>OT Velos</td><td>10</td><td>4527</td><td>427</td><td>57.8</td><td>13.5</td></tr><tr><td>L08_1</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Stage Tip<br>column</td><td>5</td><td>Q-Exactive<br>Q-Exactive<br>Q-Exactive</td><td>25</td><td>6231</td><td>1412</td><td>26.4</td><td>1.9</td></tr><tr><td>L08_2</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Stage Tip<br>column</td><td>5</td><td>Q-Exactive<br>Q-Exactive<br>Q-Exactive</td><td>25</td><td>6231</td><td>1407</td><td>56.6</td><td>4.0</td></tr><tr><td>L29</td><td>TiO2</td><td>Batch<br>Stage Tip<br>Batch<br>Batch/Phos<br>Select<br>PhosSpinTrap<br>column</td><td>24</td><td>Plus<br>Synapt G2<br>Synapt<br>5600 TTOF</td><td>16</td><td>4774</td><td>647</td><td>56.1</td><td>8.7</td></tr><tr><td>L30</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Stage Tip<br>Batch/Phos<br>Select<br>PhosSpinTrap<br>column</td><td>6</td><td>Synapt<br>5600 TTOF</td><td>10</td><td>930</td><td>437</td><td>92.6</td><td>10.0</td></tr><tr><td>L21_2</td><td>TiO2</td><td>Stage Tip<br>Batch<br>Batch/Phos<br>Select<br>PhosSpinTrap<br>column</td><td>6</td><td>Synapt<br>5600 TTOF</td><td>30</td><td>5271</td><td>623</td><td>58.5</td><td>0.7</td></tr><tr><td>L25_1</td><td>TiO2/TiO2</td><td>Batch<br>Batch/Phos<br>Select<br>PhosSpinTrap<br>column</td><td>6</td><td>5600 TTOF</td><td>25</td><td>5271</td><td>830</td><td>74.1</td><td>9.4</td></tr><tr><td>L25_2</td><td>TiO2/IMAC</td><td>Batch<br>Batch/Phos<br>Select<br>PhosSpinTrap<br>column</td><td>6</td><td>5600 TTOF</td><td>25</td><td>5271</td><td>830</td><td>74.1</td><td>8.9</td></tr><tr><td>L17</td><td>IMAC</td><td>Fe<br>Phos Select<br>IMAC</td><td>6</td><td>OT Velos<br>OT Velos<br>OT Velos<br>Orbitrap XL<br>OT Fusion<br>Lumos<br>OT Fusion<br>Lumos</td><td>30</td><td>7653</td><td>667</td><td>175.2</td><td>26.3</td></tr><tr><td>L13_3</td><td>IMAC</td><td>Phos Select<br>IMAC</td><td>6</td><td>OT Velos<br>OT Velos<br>Orbitrap XL<br>OT Fusion<br>Lumos<br>OT Fusion<br>Lumos</td><td>20</td><td>5299</td><td>476</td><td>39.1</td><td>8.2</td></tr><tr><td>L13_2</td><td>IMAC</td><td>Phos Select<br>IMAC</td><td>6</td><td>OT Velos<br>Orbitrap XL<br>OT Fusion<br>Lumos<br>OT Fusion<br>Lumos</td><td>20</td><td>5299</td><td>408</td><td>15.9</td><td>3.9</td></tr><tr><td>L23_4</td><td>IMAC</td><td>Phos Select<br>IMAC</td><td>6</td><td>Orbitrap XL<br>OT Fusion<br>Lumos<br>OT Fusion<br>Lumos</td><td>10</td><td>366</td><td>20.4</td><td>5.6</td><td>11.2</td></tr><tr><td>L23_6</td><td>IMAC</td><td>Phos Select<br>IMAC</td><td>6</td><td>Orbitrap XL<br>OT Fusion<br>Lumos<br>OT Fusion<br>Lumos</td><td>20</td><td>12,402</td><td>2114</td><td>236.5</td><td>11.2</td></tr><tr><td>L23_5</td><td>IMAC</td><td>Phos Select<br>IMAC</td><td>6</td><td>Orbitrap XL<br>OT Fusion<br>Lumos<br>OT Fusion<br>Lumos</td><td>2</td><td>10,181</td><td>972</td><td>110.8</td><td>11.4</td></tr></tbody></table>

1- Number of phosphopeptides identified in the enriched sample. Average of triplicate analysis, Std. Dev. and %CV shown when available.

enrichment experiment.

In conclusion, the use of different protocols, instruments and operators provides a wide scenario of experimental conditions that is optimal to prove the suitability of the reference material here described for inter- and intra-lab protocol benchmarking, indicating strengths, weaknesses, and guidance for optimization (Stage-Tip vs batch, sample/medium

ratio). Overall, we propose that the use of a standardized reference material in a multi-lab study is a useful resource for technology testing as has been extensively demonstrated [7–10] and provide excellent references to set up protocols and rank the performance of individual labs, contributing to the democratization of sophisticated proteomics pipelines under standardized conditions. We think that the results here
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Results of the analysis of PME11 samples reported by the different laboratories participating in the study. A) Number of phosphopeptides from each analysis. Each bar represents the average number reported, the error bars being the standard deviation of triplicate analysis performed in the same laboratory (when available). Columns are colored according to the MS instrument used for the analysis, as indicated in the legend. Results are grouped by the type of affinity enrichment used (TiO₂, IMAC). L25-2 corresponds to a two step sequential enrichment TiO₂-IMAC. The black points indicate the corrected number of phosphopeptides weighed by instrument performance (see text). B) Box and whisker plot summarizing the raw and weighed number of phosphopeptides data. C) Selectivity of the phosphopeptide enrichment measured as the % of phosphopeptides in the enriched sample. Results are shown in the same order as in Fig. 1A.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Results of the identification of the Phosphomix peptide standards. Frequency of the identification of each of the phosphomix standards in the analysis performed by all the laboratories, for each of the three standard samples, containing different amounts of the standards: A1 (100), A2 (250), A3 (500) fmol/125 µg of initial yeast digest.</figcaption></figure>

described demonstrate that the standard proposed in this study is a suitable reference material for the assessment and optimization of phosphoproteomic analysis and certainly provide valuable information to dig deeper into the pros and cons of phosphoproteomics workflows.

## Data availability

No

## Acknowledgements

We thank Kevin Ray from MilliporeSigma, Saint Louis, MO, for fruitful discussions on the study design, and MilliporeSigma for kindly providing the Phosphomix standards used in the study.

ProteoRed, PRB3 is supported by grant PT17/0019/0001, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jprot.2021.104409.

## References

[1] M.A. Jünger, R. Aebersold, Mass spectrometry-driven phosphoproteomics: patterning the systems biology mosaic, Wiley Interdiscip. Rev. Dev. Biol. 3 (1) (2014 Jan-Feb) 83–112, https://doi.org/10.1002/wdev.121. Epub 2013 Jul 2, 24902836.

[2] J.V. Olsen, M. Mann, Status of large-scale analysis of post-translational modifications by mass spectrometry, Mol. Cell. Proteomics 12 (12) (2013 Dec), https://doi.org/10.1074/mcp.O113.034181, 3444–52. Epub 2013 Nov 1, 24187339. PMC3861698.

[3] W. Qiu, C.A. Evans, A. Landels, T.K. Pham, P.C. Wright, Phosphopeptide enrichment for phosphoproteomic analysis - a tutorial and review of novel materials, Anal. Chim. Acta 1129 (2020 Sep 8) 158–180, https://doi.org/10.1016/j.aca.2020.04.053. Epub 2020 Apr 28, 32891386.

[4] M.R. Hoopmann, U. Kusebauch, M. Palmblad, N. Bandeira, D.D. Shteynberg, L. He, B. Xia, S.H. Stoychev, G.S. Omenn, S.T. Weintraub, R.L. Moritz, Insights from the first phosphopeptide challenge of the MS resource pillar of the HUPO human proteome project, J. Proteome Res. 19 (12) (2020 Dec 4) 4754–4765, https://doi.org/10.1021/acs.jproteome.0c00648. Epub 2020 Nov 9. PMID: 33166149; PMCID: PMC8204901.

[5] X. Yue, A. Schunter, A.B. Hummon, Comparing multistep immobilized metal affinity chromatography and multistep TiO2 methods for phosphopeptide enrichment, Anal. Chem. 87 (17) (2015 Sep 1) 8837–8844, https://doi.org/10.1021/acs.analchem.5b01833. Epub 2015 Aug 11, 26237447. PMC4766865.

[6] T.E. Thingholm, M.R. Larsen, Sequential elution from IMAC (SIMAC): an efficient method for enrichment and separation of mono- and multi-phosphorylated peptides, Methods Mol. Biol. 1355 (2016) 147–160, https://doi.org/10.1007/978-1-4939-3049-4_10. 26584924.

[7] V. Vialas, N. Colomé-Calls, J. Abian, K. Aloria, G. Alvarez-Llamas, O. Antúnez, J. M. Arizmendi, M. Azkargorta, S. Barceló-Batllori, M.G. Barderas, F. Blanco, J. I. Casal, V. Casas, C. de la Torre, E. Chicano-Gálvez, F. Elortza, G. Espadas, J. M. Estanyol, J. Fernandez-Irigoyen, P. Fernandez-Puente, M.J. Fidalgo, M. Fuentes, M. Gay, C. Gil, A. Hainard, M.L. Hernaez, N. Ibarrola, A.T. Kopylov, A. Lario, J. A. Lopez, M. López-Lucendo, M. Marcilla, A. Marina-Ramírez, G. Marko-Varga, L. Martín, M.I. Mora, E. Morato-López, J. Munoz, M.A. Odena, E. de Oliveira, I. Orera, I. Ortea, C. Pasquarello, K.B. Ray, M. Rezeli, I. Ruppen, E. Sabidó, M.M. S. Del Pino, J. Sancho, E. Santamaría, J. Vazquez, M. Vilaseca, F. Vivanco, J. J. Walters, V.G. Zgoda, F.J. Corrales, F. Canals, A. Paradela, A multicenter study to evaluate the use of relative retention times in targeted proteomics, J. Proteome 152 (2017 Jan 30) 138–149, https://doi.org/10.1016/j.jprot.2016.10.014. Epub 2016 Oct 29, 27989941.

[8] A. Campos, R. Díaz, S. Martínez-Bartolomé, J. Sierra, O. Gallardo, E. Sabidó, M. López-Lucendo, J. Ignacio Casal, C. Pasquarello, A. Scherl, C. Chiva, E. Borras, A. Odena, F. Elortza, M. Azkargorta, N. Ibarrola, F. Canals, J.P. Albar, E. Oliveira, Multicenter experiment for quality control of peptide-centric LC-MS/MS analysis - A longitudinal performance assessment with nLC coupled to orbitrap MS analyzers, J. Proteome 127 (Pt B) (2015 Sep 8) 264–274, https://doi.org/10.1016/j.jprot.2015.05.012. Epub 2015 May 14, 25982386.

[9] J.P. Albar, F. Canals, Standardization and quality control in proteomics, J. Proteome 95 (2013 Dec 16) 1–2, https://doi.org/10.1016/j.jprot.2013.11.002. 24275454.

[10] S.M. Mische, N.C. Fisher, S.M. Meyn, K. Sol-Church, R.L. Hegstad-Davies, F. Weis Garcia, M. Adams, J.M. Ashton, K.M. Delventhal, J.A. Dragon, L. Holmes, P. Jagtap, K.E. Kubow, C.E. Mason, M. Palmblad, B.C. Searle, C.W. Turck, K. L. Knudson, A review of the scientific rigor, reproducibility, and transparency studies conducted by the ABRF research groups, J. Biomol. Tech. 31 (1) (2020 Apr) 11–26, https://doi.org/10.7171/jbt.20-3101-003. PMID: 31969795; PMCID: PMC6959150.