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. 2023 Aug 18;22(9):100636. doi: 10.1016/j.mcpro.2023.100636

Table 1.

Mass spectrometry–based approaches for probing the secretome

Secretome approach Turn-around time Required number of cells Compatible with FCS Special reagents Strengths and disadvantages References
Serum-free ∼2 days ∼105 No Phenol-red–free basal medium
  • -

    ease of implementation, no requirement of elaborate and sophisticated enrichment techniques

  • -

    allows testing of multiple biological conditions in a short period of time

  • -

    easily scalable from dishes to multiwell cell culture plates

  • -

    versatile, compatible with different cell numbers, sample preparation methods (TCA precipitation, FASP, SP3), and SILAC labeling

  • -

    Facilitates the analysis of cell surface shedding and unconventional secretion events

  • -

    requires shift to serum-free culture conditions

  • -

    only partly suitable for tracking of transcriptional dependent processes

  • -

    serum-free conditions may affect cell viability and responsiveness

  • -

    activation of cells with proteins or ligands that require serum can be difficult

  • -

    only partly suitable for primary cells

  • -

    requires optimization of medium supplements

 (4, 5, 18, 25, 105, 106, 131, 132, 133)
AHA labeling ∼3–4 days ∼ 106 Yes Azidohomoalanine
  • -

    Compatible with serum, thus also suitable for primary cells

  • -

    Facilitates the analysis of cell surface shedding

 (37, 115, 134, 136, 138)
SILAC amino acids
  • -

    Allows stringent washing steps to remove serum contaminants

  • -

    Labeling allows distinction between cell derived and serum proteins
Nonstandard medium
formulation (no methionine)
  • -

    allows the analysis of cell surface shedding and unconventional secretion events
  • -

    compatible with SILAC labeling

Alkyne-agarose beads
  • -

    Potentially cytotoxic, demands a precise optimization of cell culture conditions and pulsing times
  • -

    methionine depletion step can disturb cells

  • -

    Only newly synthesized proteins can be analyzed, not suitable to analyze the secretion of proteins which are stored in secretory granules

  • -

    Limited in throughput due to more extensive sample work up compared to serum-free approaches

  • -

    copper-CLICK-chemistry-based pulldown requires long reaction times of approximately 18 h

  • -

    Requires high cell numbers and nonstandard media formulations without methionine

  • -

    vulnerable to perturbations of the cellular translation machinery

  • -

    click reaction can be influenced by the complexity of the sample matrix (plasma protein binding of reagents)

Azidosugar labeling (SPECS/hiSPECS) ∼4 days ∼ 106 Yes N-azidoacetyl mannosamine-tetraacylated
  • -

    Compatible with serum, thus also suitable for primary cells

  • -

    Facilitates the analysis of cell surface shedding

  • -

    Allows stringent washing steps to remove serum contaminants

  • -

    compatible with SILAC labeling

 (24, 137, 139)
DBCO-alkyne beads
  • -

    Limited in throughput due to extensive sample workup
  • -

    Requires 48 h treatment with ManNAz to label proteins

  • -

    Requires high cell numbers

  • -

    Only glycosylated proteins can be analyzed

  • -

    Dependent on a functional protein-glycosylation machinery

  • -

    unconventional secretion events of nonglycosylated proteins are missed

Proximity labeling >> 7 days, including cell line generation ∼105 Yes Plasmids or lentiviral constructs
  • -

    Compatible with serum

  • -

    Compatible in vivo and with primary cells

  • -

    Allows investigation of organ and cell type–specific protein secretion in live animals

 (122, 124, 125)
Biotin
  • -

    High temporal resolution due to fast labeling kinetics
  • -

    Requires cloning and transfection of cells or adenoviral delivery

  • -

    Labor intensive and time consuming

  • -Overexpression can lead to artificial high protein abundances and mislocalizations

FCS, fetal calf serum.