MALDI Sample Preparation at
High Throughput for Mass Spec Screening

Overcoming bottlenecks in the MALDI sample prep workflow

Fortunately, high throughput MALDI sample preparation is achievable with the right tools and technologies integrated into the workflow.  If you are looking to automate MALDI MS sample prep for HTS/uHTS applications, your system should be designed to include the following capabilities:

1536-well MALDI spotting

Consistently creating spots on a 1536-target plate is best achieved with a 1536 pipetting head that accurately delivers as little as 0.1 µL fluid across the target plate. The pipettor head must be precisely aligned to the target plate to ensure colocation/overlap of the matrix and sample target spots within a plate, as well as consistency in spot position across the entire batch of target plates.

Fixed tips are highly recommended as they are better for maintaining a precise position in the Z direction, which is very important when dispensing sub-microliter volumes using the tip touch method.

Fixed ceramic tips also have the advantage of being well-suited to perform on-target washing, as they can transfer larger volumes of wash buffer compared to a traditional pin tool.

Active MALDI spot drying

Perhaps as important as depositing and washing the MALDI spot is the drying process. Implementing an active spot drying process, comprising gentle heating and vacuum, provides more control of the resulting spot quality and makes the timing predictable.  On the other hand, allowing spots to dry naturally takes considerable time and can add variability to both your dry time and resulting data. 

Spot quality is an important contributor to achieving high precision in your data. Desorption of sample anywhere within the spot area should yield the same quantity of sample; without a uniform distribution of material across the spot, depending on exactly where the desorption takes place, there could be different amounts of sample desorbed, causing variability in quantitation.

MALDI spots actively dried by gentle heat and vacuum have a more uniform distribution of sample and matrix across the spot (Figure 1). By contrast, spots dried naturally tend to have an uneven distribution of sample across the spot.

Compare air vs. active dried MALDI spots for different matrices >

The Drug Discovery Sciences group at Boehringer Ingelheim Pharma used active drying to achieve high spot diameter precision, with a CV of <5% across the plate (Winter et al., 2018).

An additional benefit of actively drying MALDI spots is a rapid and consistent cycle time. An active drying process can keep pace with the pipetting step and be scheduled into the sample prep workflow.

Unattended operation of the MALDI-TOF sample preparation

There are two key requirements for unattended operation of an automated MALDI spotting system:

1. The system must be stocked with all reagents and consumables/labware needed.
2. The system must be able to spot as many as 100 plates without re-calibration of the pipetting deck or head.

Simon et al. (2021) set up a uHTS screen to generate upwards of one million MALDI-ToF samples and data points in a 24-hour day. To prepare target plates at that pace, all samples as well as consumables (plates, tips, and buffers) were loaded into and accessible to the pipetting deck on schedule throughout the run.

Their MALDI-TOF sample preparation system included a circulating reservoir to prevent crystallization of the matrix solution, as well as large fluid reservoirs to hold buffer for washing target spots as well as washing the fixed tips between samples and plates.

The automated MALDI sample prep platform can spot dozens of target plates without re-calibration or re-alignment more than once per day. A 1536 ceramic tip magazine accurately dispenses volumes of 0.1 to 1 µL to generate high-quality MALDI spots across hundreds of target plates.

Analytik Jena has developed systems, workflows, and technologies to automate uHTS MALDI sample prep. Learn more about Analytik Jena's MALDI application.