# Simplifying the Preparation of Pollen Grains for MALDI-TOF MS Classification

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## Abstract

**:**

## 1. Introduction

## 2. Results

_{2}O (1.25% TFA) as the matrix solvent and without any additional extraction steps (Figure 1a).

## 3. Discussion

## 4. Materials and Methods

#### 4.1. Materials

#### 4.2. Sample Preparation

- One microliter of HCCA matrix (10 mg of α-cyano-4-hydroxycinnamic acid diluted in 1 mL 1:1 acetonitrile/water (v/v) and 1.25% trifluoroacetic acid) was spotted onto the pollen grains.
- Pollen grains were deposited on the target, which was kept for 3 min in a glass box over 98% formic acid (gas phase extraction). Afterwards, 1 µL of the matrix solution (see 1) was deposited.
- One microliter of formic acid (98%) was pipetted onto the pollen grains. After drying at room temperature, 1 μL of the matrix solution (see 1) was added.

#### 4.3. Data Acquisition

#### 4.4. Data Analysis

## 5. Conclusions

_{2}O (1.25% TFA) served as matrix solvent, and no further extraction steps were conducted. The sample preparation (h in Figure 1 and Figure A1, Figure A2 and Figure A3) was pursued directly on the steel target, with gaseous formic acid extraction and TFA in the matrix solution. However, preparation (a in Figure 1 and Figure A1, Figure A2 and Figure A3) is by far the simplest sample preparation procedure; the pollen grains can be fixed easily on the conductive tape, and no further extraction steps must follow. Therefore, we suggest this procedure to establish a preoperatively simple and reliable routine method for pollen analysis based on MALDI-TOF MS. Future work will focus on the improvement of the sensitivity of this technique and the combination of MALDI imaging with multivariate data analysis.

## Acknowledgments

## Author Contributions

## Conflicts of Interest

## Appendix A

**Figure A1.**Averaged MALDI-TOF mass spectra (n = 5) of Betula tatewakiana pollen grains: measured on conductive tape with TFA in the matrix solution (

**a**–

**c**), on conductive tape without TFA in the matrix solution (

**d**–

**f**), on the steel target (MTP 384) with TFA in the matrix solution (

**g**–

**i**) and on the steel target without TFA in the matrix solution (

**j**–

**l**) (in the rows: extraction procedure).

**Figure A2.**Averaged MALDI-TOF mass spectra (n = 5) of Corylus avellana pollen grains: measured on conductive tape with TFA in the matrix solution (

**a**–

**c**), on conductive tape without TFA in the matrix solution (

**d**–

**f**), on the steel target (MTP 384) with TFA in the matrix solution (

**g**–

**i**) and on the steel target without TFA in the matrix solution (

**j**–

**l**) (in the rows: extraction procedure).

**Figure A3.**Averaged MALDI-TOF mass spectra (n = 5) of Pinus sylvestris pollen grains: measured on conductive tape with TFA in the matrix solution (

**a**–

**c**), on conductive tape without TFA in the matrix solution (

**d**–

**f**), on the steel target (MTP 384) with TFA in the matrix solution (

**g**–

**i**) and on the steel target without TFA in the matrix solution (

**j**–

**l**) (in the rows: extraction procedure).

**Figure A4.**Scores and variances of the second and third principal component (PC) of the PCA of MALDI MS spectra from four pollen species (Corylus avellana, Alnus cordata, Pinus sylvestris, Betula tatewakiana) for different sample preparation techniques (rows: variation in extraction procedure ((

**a**,

**d**,

**g**,

**j**): without additional extraction); (

**b**,

**e**,

**h**,

**k**): gaseous extraction); (

**c**,

**f**,

**i**,

**l**): droplet extraction); columns: variation in target material and matrix solution ((

**a**–

**c**): conductive tape with trifluoroacetic acid in the matrix solution); (

**d**–

**f**): CT without TFA in the matrix solution); (

**g**–

**i**): steel target, matrix with TFA); and (

**j**–

**l**): steel target, matrix without TFA)). Data pre-treatment is explained in the methods Section 4.4.

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**Figure 1.**Averaged matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectra (n = 5) of Alnus cordata pollen grains: measured on conductive tape with trifluoroacetic acid (TFA) in the matrix solution (

**a**–

**c**), on conductive tape without TFA in the matrix solution (

**d**–

**f**), on the steel target (microtiter plate MTP 384) with TFA in the matrix solution (

**g**–

**i**) and on the steel target without TFA in the matrix solution (

**j**–

**l**) (in the rows: extraction procedure).

**Figure 2.**Scores and variances of the first and second principal components (PC) of the PCA (principal component analysis) of MALDI MS spectra from four pollen species (Corylus avellana, Alnus cordata, Pinus sylvestris, Betula tatewakiana) for different sample preparation techniques (rows: variation in extraction procedure ((

**a**,

**d**,

**g**,

**j**): without additional extraction); (

**b**,

**e**,

**h**,

**k**): gaseous extraction); (

**c**,

**f**,

**i**,

**l**): droplet extraction)); columns: variation in target material and matrix solution ((

**a**–

**c**): conductive tape with trifluoroacetic acid in the matrix solution); (

**d**–

**f**): CT without TFA in the matrix solution); (

**g**–

**i**): steel target, matrix with TFA); and (

**j**–

**l**): steel target, matrix without TFA)). Data pre-treatment is explained in the methods Section 4.4.

**Figure 3.**Loadings and variances (in brackets) of the first PC (top, black), the second PC (middle, gray) and the third PC (bottom, light gray) of the respective PCAs (rows: variation in extraction procedure ((

**a**,

**d**,

**g**,

**j**): without additional extraction); (

**b**,

**e**,

**h**,

**k**): gaseous extraction); (

**c**,

**f**,

**i**,

**l**): droplet extraction)); columns: variation in target material and matrix solution ((

**a**–

**c**): conductive tape with trifluoroacetic acid in the matrix solution); (

**d**–

**f**): CT without TFA in the matrix solution); (

**g**–

**i**): steel target, matrix with TFA); and (

**j**–

**l**): steel target, matrix without TFA)), for better comparison, graph (

**a**,

**h**) are highlighted.

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**MDPI and ACS Style**

Lauer, F.; Seifert, S.; Kneipp, J.; Weidner, S.M.
Simplifying the Preparation of Pollen Grains for MALDI-TOF MS Classification. *Int. J. Mol. Sci.* **2017**, *18*, 543.
https://doi.org/10.3390/ijms18030543

**AMA Style**

Lauer F, Seifert S, Kneipp J, Weidner SM.
Simplifying the Preparation of Pollen Grains for MALDI-TOF MS Classification. *International Journal of Molecular Sciences*. 2017; 18(3):543.
https://doi.org/10.3390/ijms18030543

**Chicago/Turabian Style**

Lauer, Franziska, Stephan Seifert, Janina Kneipp, and Steffen M. Weidner.
2017. "Simplifying the Preparation of Pollen Grains for MALDI-TOF MS Classification" *International Journal of Molecular Sciences* 18, no. 3: 543.
https://doi.org/10.3390/ijms18030543