Table 3.

Comparison of different LC and MS methods with their advantages and limitations for HCP analysis.

LC Separation Methods	Advantages	Limitations
1D LC-MS/MS (analytical flow)	well established, very robust, analytical flow already implemented in regulated laboratories	less sensitive compared to micro/nano flow
2D LC-MS/MS	wide dynamic range, enhanced peptide selectivity, higher sensitivity	labor intensive and time consuming, large quantity of starting material required
Long columns/gradients	enhanced peptide separation, higher sensitivity	reduced sample throughput per day
Micro/nano flow	enhanced sensitivity, reduced solvent consumption and source contamination, superior for limited sample amounts	longer time for method development, comprised robustness
MS Acquisition Methods	Advantages	Limitations
DDA	gold standard for identification, well established	less accurate quantitative information (only MS1 or spectral counting)
DIA	generic platform method for unknown proteins, comprehensive data set allows reprocessing, label-free quantification	method optimization requires more expertise, challenging deconvolution, spectral library required: experimentally or predicted, robust data analysis requires statistical expertise
Targeted MRM/PRM	higher signal and increased S/N compared to DDA/DIA, increased sensitivity, accurate, selective	extensive method development for each target protein, requires prior knowledge, limited multiplexing of target proteins, requires production of reference peptides