README file
Data Set Title: “Data for the application of a robust MALDI mass spectrometry approach for bee pollen investigation”
Data Set Author/s: 
Chiara Braglia^a: https://orcid.org/0000-0002-1637-896X
Daniele Alberoni^a: https://orcid.org/0000-0002-2394-2880
Alessandra Giacomelli^b: https://orcid.org/0000-0002-6012-2937
Michel Bocquet^c:  https://orcid.org/0000-0002-1584-1481
Diana Di Gioia^a: https://orcid.org/0000-0002-0181-1572
Philippe Bulet^d,e: https://orcid.org/0000-0001-9016-265X 

^a Dipartimento di Scienze e Tecnologie Agro-Alimentari (DISTAL), University of Bologna, Viale Fanin 42, 40127, Bologna, Italy;  
 
^b Unione Nazionale Associazioni Apicoltori Italiani (UNA API), Via Paolo Boselli 35, Firenze, Italy

^c Apimedia, 82 Route de Proméry, Pringy, 74370 Annecy, France 

^d CR, University Grenoble Alpes, IAB Inserm 1209, CNRS UMR5309, 38000 Grenoble, France
^e Plateforme BioPark of Archamps, 74160 Archamps, France

Data Set Contributor/s: 
Michel Bocquet:  https://orcid.org/0000-0002-1584-1481
Chiara Braglia: https://orcid.org/0000-0002-1637-896X
Philippe Bulet: https://orcid.org/0000-0001-9016-265X 

Data Set Contact Person/s: 
Daniele Alberoni: daniele.alberoni@unibo.it

Data Set License: this data set is distributed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.

Publication Year: 2024
This study received the financial support of CRT1-Ambiente CUP code J18H23000520001 managed by UNAAPI- Unione Nazionale Associazioni Apicoltori Italiani, and the project SWEET2 - Italian Ministry of Agriculture CUP code J38H23001110001 managed by the University of Bologna. This study was carried out within the Agritech National Research Center and received funding from the European Union Next-GenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 – D.D. 1032 17/06/2022, CN00000022). 

Data set Contents
The data set consists of:
 •	1 README file 
“MALDI_MS_README.rtf”
 •	1 archive file saved in /.zip format
MALDI_MS_Machine_Output_Raw_Data.zip
This archive contains the MALDI MS spectra, and raw data. The files are in .fid and .1rc format 

The archive file .fid and .1rc  be opened only with the ClinProTools software (BRUKER software) or with open source software such as Mass-Up (https://www.sing-group.org/massup/ ) or OpenChrom (https://openchrom.net/ )

 •	1 tabular quantitative file saved in /.xlsx format 
“Raw_Data_MALDI-MS_in-pollen-samples_07062024”
This file report all the exported data from the files .fid and .1rc contained in the MALDI_MS_Machine_Output_Raw_Data.zip

The excel file contains the following columns:
 o	Rank: machine output
 o	File Name: it is the raw data complete name. This name connects to the zip file “MALDI_MS_Machine_Output_Raw_Data.zip”, and the files contained within the zip file. E.g.: the file name “Spectrum: D:\Data\2023\231026\A396353CBRA02\0_A2\1\1SLin” connects to zip folder at the file “MALDI_MS_Machine_Output_Raw_Data.zip\Polline_famiglie\Cistaceae\ of the peak”
 o	Run: for every sample it is the same. 
 o	Pollen Number: internal code identifying the pollen sample.
 o	RefMassTXT: m/z automatic rounding off
 o	mz: Effective detected by MALDI TOF m/z
 o	Time: time of detection
 o	Intens: Intensity of the peak 
 o	SN: Signal to noise of the peak
 o	Res: Resolution of the peak
 o	Area: Area of the peak
 o	RelIntens: Relative intensity of the peak
 o	FWHM: full width at half-maximum intensity of the peak
 o	Spectra: internal number of the spectra file.  eg: A396353CBRA02
 o	Color: corbicular pollen color
 o	PollenRegion: Region of collection of the corbicular pollen 
 o	SpeciesPollen: Botanical specie of the corbicular pollen
 o	CollectionDate: Date of collection of corbicular pollen
 o	CollectionPeriod: Period of collection of corbicular pollen (first two weeks of the month or second two weeks of the month)
 o	ExperimentNb: number of the experiment (in our case just 1)
 o	Family: corbicular pollen botanical family
 o	Genera: corbicular pollen botanical genera
 o	Specie: corbicular pollen botanical specie

Data set Documentation
Abstract
The dataset contains results on honeybee pollen extracted and analyzed with MALDI MS. 
Our experiment involved honeybee pollen grains from five different Italian regions (Campania, Sardinia, Sicily, South Tirol and Tuscany) through the spring 2023. Pollen sample were collected from the honeybee colonies with standard pollen traps every two weeks for four months. The generated dataset concern different pollen extraction methods. In order to define the best experimental conditions to record robust and the most representative and distinguishable spectra for a pollen species, different conditions of extraction were tested, four different  solutions: (a) 2M acetic acid 2M (2M AA) and 50% acetonitrile (50% ACN); (b) AA 2M; (c) 2% ACN and 0.1% trifluoroacetic acid (0.1% TFA); and (d) a solution of 1% TFA) and two mechanical extraction methods (stirring and ultrasonication). Moreover, 10-fold serial dilutions of the crude extracted material from the different bee pollen balls were evaluated (10, 100 and 1,000 times) to determine the most appropriate sample to matrix ratio.

Materials and methods
Pollens collection and identification
Corbicular pollen balls were collected in five Italian regions characterized by different latitude, climate conditions and plant resources (Campania, Sardegna, Sicilia, Trentino-Alto Adige, and Toscana) using traditional pollen traps. The collected polyfloral pollen was divided into monofloral pollen samples and identified with the help of the international database PalDat - Palynological Database.

MALDI-MS 
1. Pollen preparation
Single pollen balls were processed with four different extractions solutions using different solvents dissolved, when necessary, in ultrapure water. The four tested solvent solutions were: (1) 2M pure acetic acid (AA) additioned with 50% acetonitrile (ACN); (2) 2M pure AA; (3) a mixture of 2% ACN and 0.1% trifluoroacetic acid (TFA); (4) 1% TFA. A total volume of 50µL of each solvent solution was added to a single pollen ball. 
For each pollen extracted with solvents, two different disruption methods were tested: a sonication cycle of 15 minutes at 60Hz and a shaken approach at 800 rpm for 1 hour at 4°C. The obtained extracts were diluted 10, 100 and 1000 times in 2% ACN in 0.1 % TFA. The matrix used was alpha-cyano-4-hydroxycinnamic acid matrix (4-CHCA).

2. Data acquisition
MALDI molecular mass fingerprints (MFPs) were acquired in a positive ion linear mode using an AutoFlex III - Smartbeam® mass spectrometer (Bruker Daltonics, Germany) (Houdelet et al., 2021; Ajdi et al., 2023). An external mass spectrometer calibration was achieved using the PepMix and ProtMix (Bruker Daltonics) standard calibration kits covering the dynamic range of 700 and 8560 Da. 

3. Data processing
MALDI-MS data were visualized with FlexAnalysis v3.4 Software (Bruker Daltonics, Germany) and post-processed and analyzed (PCA and modeling) using ClinProTools™v2.2 Software (Bruker Daltonics, Germany).

File specifics
The archive file .fid and .1rc  be opened only with the ClinProTools software (BRUKER software) or with open source software such as Mass-Up (https://www.sing-group.org/massup/ ) or OpenChrom (https://openchrom.net/ )
The Excel file, can be opened with Microsoft Excel or with Open Office softwares